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Title:  Peptides

United States Patent:  6,849,601

Issued:  February 1, 2005

Inventors:  Lewis; Richard James (Woolloongabba, AU); Alewood; Paul Francis (Moggill, AU); Sharpe; Iain Andrew (Taringa, AU)

Assignee:  The University of Queensland (St. Lucia, AU)

Appl. No.:  806376

Filed:  October 18, 2001

PCT Filed:  October 1, 1999

PCT NO:  PCT/AU99/00843

371 Date:  October 18, 2001

102(e) Date:  October 18, 2001

PCT PUB.NO.:  WO00/20443

PCT PUB. Date:  April 13, 2000

Abstract

The present invention relates to novel peptides and derivatives thereof useful as selective .alpha.1 -adrenoceptor antagonists which ability of inhibiting the agonist action of agonist noradrenaline on the .alpha.1 -adrenoceptor is greater than their ability of inhibiting the action of agonist noradrenaline on the other .alpha.-adrenoceptors. The invention also relates to pharmaceutical compositions comprising these peptides.

Description of the Invention

The present invention relates to novel peptides and derivatives thereof useful as selective .alpha.1 -adrenoceptor antagonists. 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 selective .beta.1 -adrenoceptor antagonist activity and the use of these peptides in the prophylaxis or treatment of conditions such as but not limited to urinary or cardiovascular conditions.

The marine snails of the genus Conus (cone snails) use a sophisticated biochemical strategy to capture their prey. As predators of either fish, worms or other molluscs, the cone snails inject their prey with venom containing a cocktail of small bioactive peptides. These toxin molecules, which are referred to as conotoxins, interfere with neurotransmission by targeting a variety of 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 to-conotoxin class of peptides target and block voltage-sensitive Ca2+ -channels inhibiting neurotransmitter release. The .alpha.-conotoxins and .psi.-conotoxins target and block nicotinic acetylcholine (ACh) receptors, causing ganglionic and neuromuscular blockade. Peptides of the .mu.conotoxin class act to block voltage-sensitive Na+ -channels, inhibiting muscle and nerve action potentials. The .delta.-conotoxins target and delay the inactivation of voltage-sensitive Na+ -channels, enhancing neuronal excitability. The .kappa.-conotoxin class of peptides target and block voltage-sensitive K+ -channels, and these may also cause enhanced neuronal excitability. The conopressins are vasopressin receptor antagonists and the conantokins are N-methyl-D-aspartate (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 antagonises the 5HT3 receptor, have been described.

It has now been found that a new class of conotoxin exists, hereafter referred to as the .rho.-conotoxin class, which are characterised by having .alpha.1 -adrenoceptor antagonist activity. .alpha.1 -Adrenoceptors play important roles in many physiological and pathophysiological processes of the cardiovascular and urogenital systems, including myocardial inotropy and chronotropy, cardiac hypertrophy and arrhythmias, vasoconstriction, smooth muscle contraction and prostate disease. .alpha.1 -adrenoceptor antagonist drugs are of use as both tools for basic research and as therapeutic agents.

U.S. Pat. No. 5,620,993 (Patane et al) describes some of the known functions of adrenergic receptors of the .alpha.1 -subtype, as well as some of the known pharmacological agents which bind to them. The peptides of the present invention are the first peptides reported to have .alpha.1 -adrenoceptor antagonist activity. Further .rho.-conotoxin peptides act non-competitively to inhibit noradrenaline action. Thus, it appears that .rho.-conotoxins act at a site distinct from the site of noradrenaline activation and distinct from the site of action of traditional .alpha.-adrenorecptor antagonists such as prazosin.

Accordingly in one aspect of the present invention there is provided an isolated, synthetic or recombinant .rho.-conotoxin peptide having selective .beta.1 -adrenoceptor antagonist activity.

The .rho.-conotoxin peptide may be a naturally occurring peptide isolated from a cone snail, or a derivative thereof.

Preferably the .rho.-conotoxin peptide is .rho.-TIA or a derivative thereof. .rho.-TIA may be isolated from the venom duct of the fish hunting cone snail Conus tulipa. It is a peptide comprising 19 amino acids and contains two disulphide bonds. The amino acid sequence of .rho.-TIA is as follows.

FNWRCCLIPACRRNHKKFC

(SEQ ID NO:1)

The C-terminus may be a free acid or amidated.

As used herein the term "selective", unless the context requires otherwise, means that the ability of the peptide to act as an antagonist of an .beta.1 -adrenoceptor is considerably greater than its ability to act as an antagonist of other .alpha.-adrenoceptors. Preferably the activity at other .alpha.-adrenoceptors is negligible.

The term "derivative" as used herein in connection with naturally occurring .rho.-conotoxin peptides, such as .rho.-TIA, 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 selective .beta.1 -adrenoceptor antagonist activity do not fall within the scope of the present invention. One such inactive derivative is the truncated .rho.-TIA as shown below:

CCLIPACRRNHKKFC

(SEQ ID NO: 2)

Studies of C-terminal truncation of .rho.-TIA have indicated that the residue at position 4 may be important for binding. Accordingly peptides in which the arginine residue at position 4 is retained or substituted with another amino acid with a positive charge are preferred.

It has also been found that the residues at positions 1, 2 and 3 can be substituted to modify potency and selectivity of .rho.-TIA. Such modifications include addition or substitution of one or more tyrosine residues which would allow easy labelling of .rho.-TIA derivatives for assay development.

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 ##STR1##

It is to be understood that some non-conventional amino acids may also be suitable replacements for the naturally occurring amino acids. For example ornithine, homoarginine and dimethyllysine are related to His, Arg and Lys.

Substitutions encompassed by the present invention may also be "non-conservative", in which an amino acid residue which is present in a polypeptide is substituted with an amino acid having different properties, such as naturally-occurring amino acid from a different group (eg. substituting a charged or hydrophobic amino acid with alanine), or alternatively, in which a naturally-occurring amino acid is substituted with a non-conventional amino acid.

Amino acid substitutions are typically of single residues, but may be of multiple residues, either clustered or dispersed.

Preferably, amino acid substitutions are conservative.

Additions encompass the addition of one or more naturally occurring or non-conventional amino acid residues. Deletion encompasses the deletion of one or more amino acid residues.

As stated above the present invention includes peptides in which one or more of the amino acids has undergone side chain modifications. Examples of side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NABH4 ; amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2,4,6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5-phosphate followed by reduction with NaBH4.

The guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.

The carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivitisation, for example, to a corresponding amide.

Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of 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 sulphydryl groups of cysteine with selenium equivalents such that the peptide forms a diselenium bond in place of one or more of the disulphide bonds.

Tryptophan residues may be modified by, for example, oxidation with N-bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides. Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.

Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carbethoxylation with diethylpyrocarbonate.

Proline residue may be modified by, for example, hydroxylation in the 4-position.

A list of some amino acids having modified side chains and other unnatural amino acids is shown in Table 1.

            TABLE 1
            Non-conventional
            amino acid                         Code
            .alpha.-aminobutyric acid          Abu
            .alpha.-amino-.alpha.-methylbutyrate Mgabu
            aminocyclopropane-                 Cpro
            carboxylate
            aminoisobutyric acid               Aib
            aminonorbornyl-                    Norb
            carboxylate
            cyclohexylalanine
            cyclopentylalanine                 Cpen
            D-alanine                          Dal
            D-arginine                         Darg
            D-aspartic acid                    Dasp
            D-cysteine                         Dcys
            D-glutamine                        Dgln
            D-glutamic acid                    Dglu
            D-histidine                        Dhis
            D-isoleucine                       Dile
            D-leucine                          Dleu
            D-lysine                           Dlys
            D-methionine                       Dmet
            D-ornithine                        Dorn
            D-phenylalanine                    Dphe
            D-proline                          Dpro
            D-serine                           Dser
            D-threonine                        Dthr
            D-tryptophan                       Dtrp
            D-tyrosine                         Dtyr
            D-valine                           Dval
            D-a-methylalanine                  Dmala
            D-.alpha.-methylarginine           Dmarg
            D-.alpha.-methylasparagine         Dmasn
            D-.alpha.-methylaspartate          Dmasp
            D-.alpha.-methylcysteine           Dmcys
            D-.alpha.-methylglutamine          Dmgln
            D-.alpha.-methylhistidine          Dmhis
            D-.alpha.-methylisoleucine         Dmile
            D-.alpha.-methylleucine            Dmleu
            D-.alpha.-methyllysine             Dmlys
            D-.alpha.-methylmethionine         Dmmet
            D-.alpha.-methylornithine          Dmorn
            D-.alpha.-methylphenylalanine      Dmphe
            D-.alpha.-methylproline            Dmpro
            D-.alpha.-methylserine             Dmser
            D-.alpha.-methylthreonine          Dmthr
            D-.alpha.-methyltryptophan         Dmtrp
            D-.alpha.-methyltyrosine           Dmty
            D-.alpha.-methylvaline             Dmval
            D-N-methylalanine                  Dnmala
            D-N-methylarginine                 Dnmarg
            D-N-methylasparagine               Dnmasn
            D-N-methylaspartate                Dnmasp
            D-N-methylcysteine                 Dnmcys
            D-N-methylglutamine                Dnmgln
            D-N-methylglutamate                Dnmglu
            D-N-methylhistidine                Dnmhis
            D-N-methylisoleucine               Dnmile
            D-N-methylleucine                  Dnmleu
            D-N-methyllysine                   Dnmlys
            N-methylcyclohexylalanine          Nmchexa
            D-N-methylornithine                Dnmorn
            N-methylglycine                    Nala
            N-methylaminoisobutyrate           Nmaib
            N-(1-methylpropyl)glycine          Nile
            N-(2-methylpropyl)glycine          Nleu
            D-N-methyltryptophan               Dnmtrp
            D-N-methyltyrosine                 Dnmtyr
            D-N-methylvaline                   Dnmval
            .gamma.-aminobutyric acid          Gabu
            L-t-butylglycine                   Tbug
            L-ethylglycine                     Etg
            L-homophenylalanine                Hphe
            L-.alpha.-methylarginine           Marg
            L-.alpha.-methylaspartate          Masp
            L-.alpha.-methylcysteine           Mcys
            L-.alpha.-methylglutamine          Mgln
            L-.alpha.-methylhistidine          Mhis
            L-.alpha.-methylisoleucine         Mile
            L-.alpha.-methylleucine            Mleu
            L-.alpha.-methylmethionine         Mmet
            L-.alpha.-methylnorvaline          Mnva
            L-.alpha.-methylphenylalanine      Mphe
            L-.alpha.-methylserine             Mser
            L-.alpha.-methyltryptophan         Mtrp
            L-.alpha.-methylvaline             Mval
            N-(N-(2,2-diphenylethyl)           Nnbhm
            carbamylmethylglycine
            1-carboxy-1-(2,2-diphenyl-         Nmbc
            ethylamino)cyclopropane
            L-N-methylalanine                  Nmala
            L-N-methylarginine                 Nmarg
            L-N-methylasparagine               Nmasn
            L-N-methylaspartic acid            Nmasp
            L-N-methylcysteine                 Nmcys
            L-N-methylglutamine                Nmgln
            L-N-methylglutamic acid            Nmglu
            Chexa L-N-methylhistidine          Nmhis
            L-N-methylisolleucine              Nmile
            L-N-methylleucine                  Nmleu
            L-N-methyllysine                   Nmlys
            L-N-methylmethionine               Nmmet
            L-N-methylnorleucine               Nmnle
            L-N-methylnorvaline                Nmnva
            L-N-methylornithine                Nmorn
            L-N-methylphenylalanine            Nmphe
            L-N-methylproline                  Nmpro
            L-N-methyserine                    Nmser
            L-N-methylthreonine                Nmthr
            L-N-methyltryptophan               Nmtrp
            L-N-methyltyrosine                 Nmtyr
            L-N-methylvaline                   Nmval
            L-N-methylethylglycine             Nmetg
            L-N-methyl-t-butylglycine          Nmtbug
            L-norleucine                       Nle
            L-norvaline                        Nva
            .alpha.-methyl-aminoisobutyrate    Maib
            .alpha.-methyl-.gamma.-aminobutyrate Mgabu
            .alpha.-methylcyclohexylalanine    Mchexa
            .alpha.-methylcyclopentylalanine   Mcpen
            .alpha.-methyl-.alpha.-napthylalanine Manap
            .alpha.-methylpenicillamine        Mpen
            N-(4-aminobutyl)glycine            Nglu
            N-(2-aminoethyl)glycine            Naeg
            N-(3-aminopropyl)glycine           Norn
            N-amino-.alpha.-methylbutyrate     Nmaabu
            .alpha.-napthylalanine             Anap
            N-benzylglycine                    Nphe
            N-(2-carbamylethyl)glycine         Ngln
            N-(carbamylmethyl)glycine          Nasn
            N-(2-carboxyethyl)glycine          Nglu
            N-(carboxymethyl)glycine           Nasp
            N-cyclobutylglycine                Ncbut
            N-cycloheptylglycine               Nchep
            N-cyclohexylglycine                Nchex
            N-cyclodecylglycine                Ncdec
            N-cyclododecylglycine              Ncdod
            N-cyclooctylglycine                Ncoct
            N-cyclopropylglycine               Ncpro
            N-cycloundecylglycine              Ncund
            N-(2,2-diphenylethyl)glycine       Nbhm
            N-(3,3-diphenylpropyl)glycine      Nbhe
            N-(3-guanidinopropyl)glycine       Narg
            N-(1-hydroxyethyl)glycine          Nthr
            N-(hydroxyethyl))glycine           Nser
            N-(imidazolylethyl))glycine        Nhis
            N-(3-indolylyethyl)glycine         Nhtrp
            N-methyl-.gamma.-aminobutyrate     Nmgabu
            D-N-methylmethionine               Dnmmet
            N-methylcyclopentylalanine         Nmcpen
            D-N-methylphenylalanine            Dnmphe
            D-N-methylproline                  Dnmpro
            D-N-methylserine                   Dnmser
            D-N-methylthreonine                Dnmthr
            N-(1-methylethyl)glycine           Nval
            N-methyla-napthylalanine           Nmanap
            N-methylpenicillamine              Nmpen
            N-(p-hydroxyphenyl)glycine         Nhtyr
            N-(thiomethyl)glycine              Ncys
            penicillamine                      Pen
            L-.alpha.-methylalanine            Mala
            L-.alpha.-methylasparagine         Masn
            L-.alpha.-methyl-t-butylglycine    Mtbug
            L-methylethylglycine               Metg
            L-.alpha.-methylglutamate          Mglu
            L-.alpha.-methylhomophenylalanine  Mhphe
            N-(2-methylthioethyl)glycine       Nmet
            L-.alpha.-methyllysine             Mlys
            L-.alpha.-methylnorleucine         Mnle
            L-.alpha.-methylornithine          Morn
            L-.alpha.-methylproline            Mpro
            L-.alpha.-methylthreonine          Mthr
            L-.alpha.-methyltyrosine           Mtyr
            L-N-methylhomophenylalanine        Nmhphe
            N-(N-(3,3-diphenylpropyl)          Nnbhe
            carbamylmethylglycine
            O-methyl-L-serine                  Omser
            O-methyl-L-homoserine              Omhser

These types of modifications may be important to stabilise the peptide if administered to an individual or for use as a diagnostic reagent.

Other derivatives contemplated by the present invention include a range of glycosylation variants from a completely unglycosylated molecule to a modified glycosylated molecule. Altered glycosylation patterns may result from expression of recombinant molecules in different host cells.

The .rho.-conotoxins 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.

Preferably the derivatives of naturally occurring .rho.-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.

In modification to form derivatives of naturally occurring .rho.-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.

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 .alpha.1 -adrenoceptor subtype selectivity. Non-native amino acid changes could also be included to improve potency, selectivity and/or stability.

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 TIA further modifies activity.

It is noted that .rho.-TIA is composed of a tail (residues 1-4) and two loops (residues 7-10 and 12-18), however the .rho.-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 selective .alpha.1 -adrenoceptor antagonist activity, a peptide will fall within the scope of the present invention. Preferably the peptides will have at least two cysteine residues and at lease one disulphide bond, or more preferably four cysteine residues and two disulphide bonds.

The connectivity of the disulfide bonds in these peptides may be A-C/B-D, A-D/B-C or A-B/C-D, the former being preferred for .rho.-TIA. A, B, C and D refer to the first, second, third and fourth Cys residues involved in disulphide bond formation, respectively.

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 .rho.-TIA 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 adrenoceptors 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 .rho.-TIA, 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.

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.

The .rho.-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.

References describing the synthesis of conotoxins include Sato et al, Lew et al and WO 91/07980.

The .rho.-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.

The invention further provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to sequence encoding a .rho.-conotoxin peptide as described above.

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 .rho.-conotoxin peptide.

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.

As used herein a reference to a "probe" includes reference to a primer used in amplification or a probe for use in direct hybridization.

Still another aspect of the present invention is directed to antibodies to the .rho.-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 invent ion are particularly useful as therapeutic or diagnostic agents.

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.

It may also be possible to prepae antiidiotypic antibodies using techniques known to the art. These antiidiotypic antibodies and their use as therapeutic agents represent a further aspect of the invention.

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.

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.

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.

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.

The present invention extends to such genetic constructs and to prokaryotic or eukaryotic cells comprising same.

Chimeras of .rho.-conotoxins such as .rho.-TIA, 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.

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 .rho.-TIA disulphide bonds link the first and third, and the second and fourth cysteine residues. This pattern is similar to the binding pattern observed for .alpha.-conotoxin peptides. Accordingly chimeric derivatives may be prepared by substituting a loop of a .rho.-conotoxin peptide with the loop comprising a sequence from another peptide, including .beta.-conotoxins.

The invention also includes dimers, trimers, etc. of .rho.-conotoxin peptides as well as .rho.-conotoxin peptides bound to other peptides.

Preferably the .rho.-conotoxin peptides according to the invention have 10 to 30 amino acids, more preferably 15 to 25.

The complete gene sequence for the naturally occurring .rho.-conotoxin peptides may be obtained using a combined 5' RACE and 3' RACE strategy coupled with cloning and DNA sequencing.

Although .rho.-TIA displays some sequence homology to the .alpha.-conotoxins, which are nicotinic ACh receptor blockers, .rho.-TIA (10 .mu.M) was not found to target the neuronal or muscle subtype of the nicotinic ACh receptor in assays using isolated preparations of the guinea pig ileum and the mouse phrenic nerve-hemidiaphragm.

Accordingly in a preferred aspect of the present invention the .rho.-conotoxin peptide is further characterised by lacking activity at the neuronal or muscle subtype of the nicotinic ACh receptor.

It was also found in binding studies that there is a variation in affinity of .rho.-TIA to the .alpha.1a, .alpha.1b and .alpha.1d -adrenoceptor subtypes. Accordingly in a further aspect of the invention there is provided an isolated, synthetic or recombinant .rho.-conotoxin peptide having selective .alpha.1 -antagonist activity, and having a selectivity for one .alpha.1 subtype over the other subtypes.

The .rho.-conotoxin peptides according to the present invention are selective .alpha.1 -adrenoceptor antagonists. Accordingly the invention provides the use of a .rho.-conotoxin according to the invention as a selective .beta.1 -adrenoceptor antagonist, and in the treatment or prophylaxis of diseases or conditions in relation to which antagonist activity at .alpha.1 -adrenoceptors is associated with effective treatment. Such activity in pharmacological agents is associated with efficacy 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.

Accordingly the present invention provides a method for the treatment or prophylaxis of urinary or cardiovascular conditions or diseases or mood disorders, or in 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 .rho.-conotoxin peptide having selective .alpha.1 -adrenoceptor antagonist activity.

Examples of diseases or conditions of the urinary system include benign prostatic hyperplasia and related disorders. Examples of cardiovascular diseases or conditions include arrhythmia of various regions, hypertension and coronary heart failure. Examples of mood disorders include cravings such as smoking. Examples of pain include chronic pain, neuropathic pain and inflammatory pain.

Preferably the mammal is in need of such treatment although the peptide may be administered in a prophylactic sense.

The invention also provides a composition comprising an isolated, synthetic or recombinant .rho.-conotoxin peptide having selective .beta.1 -adrenoceptor antagonist activity, and a pharmaceutically acceptable carrier or diluent.

Preferably the composition is in the form of a pharmaceutical composition.

There is also provided the use of an isolated, synthetic or recombinant .rho.-conotoxin peptide having selective .beta.1 -adrenoceptor antagonist activity in the manufacture of a medicament 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.

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.

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.

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.

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.

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.

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.

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.

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 or epidural delivery.

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.

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.

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 0.25 .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.

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.

Claim 1 of 11 Claims

What is claimed is:

1. An isolated, synthetic or recombinant .rho.-conotoxin peptide having selective .beta.1 -adrenoceptor antagonist activity whereby the ability of said peptide to inhibit the agonist action of nor-adrenaline at an .alpha.1 -adrenoceptor is greater than its ability to inhibit the agonist action of nor-adrenaline at other .alpha.-adrenoceptors.


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