Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the invention relates to relatively short peptides (termed ⁇ -conotoxins herein), about 10-30 residues in length, which are naturally available in minute amounts in the venom of the cone snails or analogous to the naturally available peptides, and which preferably include two disulfide bonds.
• the predatory cone snails have developed a unique biological strategy.
• Their venom contains relatively small peptides that are targeted to various neuromuscular receptors and may be equivalent in their pharmacological diversity to the alkaloids of plants or secondary metabolites of microorganisms.
• Many of these peptides are among the smallest nucleic acid- encoded translation products having defined conformations, and as such, they are somewhat unusual.
• Peptides in this size range normally equilibrate among many conformations. Proteins having a fixed conformation are generally much larger.
• cone snails that produce these peptides are a large genus of venomous gastropods comprising approximately 500 species. All cone snail species are predators that inject venom to capture prey, and the spectrum of animals that the genus as a whole can envenomate is broad. A wide variety of hunting strategies are used, however, every Conus species uses fundamentally the same basic pattern of envenomation.
• peptides isolated from Conus venoms have been characterized. These include the ⁇ -, ⁇ - and ⁇ -conotoxins which target nicotinic acetylcholine receptors, muscle sodium channels, and neuronal calcium channels, respectively (Olivera et al., 1985). Conopressins, which are vasopressin analogs, have also been identified (Cruz et al.. 1987). In addition, peptides named conantokins have been isolated from Conus geographus and Conus tulipa (Mena et al., 1990; Haack et al., 1990).
• the ⁇ -conotoxins are small peptides highly specific for neuromuscular junction nicotinic acetylcholine receptors (Gray et al., 1981; Marshall and Harvey, 1990; Blount et al., 1992; Jacobsen et al., 1997) or highly specific for neuronal nicotinic acetylcholine receptors (Fainzilber et al., 1994; Johnson et al., 1995; Cartier et al., 1996; Luo et al., 1998).
• the ⁇ -conotoxins with specificity for neuromuscular junction nicotinic acetylcholine receptors are used as neuromuscular blocking agents for use in conjunction with surgery, as disclosed in U.S. patent application Serial No. 09/ , filed 21 January 2000 (Attorney Docket No. 2314-178. A) and international patent application No.
• ⁇ -conotoxins with specificity for neuronal nicotinic acetylcholine receptors are used for treating disorders regulated at neuronal nicotinic acetylcholine receptors.
• disorders include, but are not limited to, cardiovascular disorders, gastric motility disorders, urinary incontinence, nicotine addiction, mood disorders (such as bipolar disorder, unipolar depression, dysthymia and seasonal effective disorder) and small cell lung carcinoma, as well as the localization of small cell lung carcinoma.
• the invention relates to relatively short peptides (termed ⁇ -conotoxins herein), about 10-30 residues in length, which are naturally available in minute amounts in the venom of the cone snails or analogous to the naturally available peptides, and which preferably include two disulfide bonds.
• ⁇ -conotoxin peptides having the general formula I:
• Xaa 8 is Ser, Thr, Asn, Ala, Gly, His, halo-His, Pro or hydroxy-Pro
• Xaa ⁇ is Thr, Ser, Ala, Asp, Asn, Pro, hydroxy- Pro, Arg, ornithine, homoarginine, Lys, N-methyl-Lys, N,N-dimethyl-Lys, N,N,N-trimethyl-Lys or any unnatural basic amino acid
• Xaa 10 is Gly, Ser, Thr, Ala, Asn, Arg, ornithine, homoarginine, Lys, N-methyl-Lys, N-N-dimethyl-Lys, N,N,N-trimethyl-Lys or any unnatural basic amino acid;
• Xaa is Gin, Leu, His, halo-His, Trp (D or L), halo-Trp, neo-Trp, Tyr, nor-Tyr, mono-halo-Tyr, di- halo-Tyr, O-sulpho-Tyr, O-phospho-Tyr, nitro-Tyr, Arg, ornithine, homoarginine, Lys, N-methyl- Lys, N-N-dimethyl-Lys, N,N,N-trimethyl-Lys, any unnatural basic amino acid or any unnatural aromatic amino acid;
• Xaa, 2 is Asn, His, halo-His, He, Leu, Val, Gin, Arg, ornithine, homoarginine, Lys, N-methyl-Lys, N,N-dimethyl-Lys, N,N,N-trimethyl-Lys or any unnatural basic amino acid;
• Xaa, 3 is des-Xaa 13 , Val, He, Leu, Arg, ornithine, homoarginine, Lys, N-methyl-Lys, N-N-dimethyl- Lys, N,N,N-trimethyl-Lys or any unnatural basic amino acid.
• the C-terminus may contain a free carboxyl group or an amide group.
• the halo is chlorine, bromine or iodine, preferably iodine for Tyr and His and preferably bromine for Trp.
• the Cys residues may be in D or L configuration and may optionally be substituted with homocysteine (D or L).
• the Tyr residues may be substituted with the 3-hydroxyl or 2-hydroxyl isomers and corresponding O-sulpho- and O-phospho- derivatives.
• the acidic amino acid residues may be substituted with any synthetic acidic bioisoteric amino acid surrogate, e.g., tetrazolyl derivatives of Gly and Ala.
• ⁇ -conotoxin peptides having the general formula II:
• the C-terminus may contain a free carboxyl group or an amide group.
• the halo is preferably bromine, chlorine or iodine, more preferably iodine for His or Tyr and bromine for Trp.
• the Cys residues may be in D or L configuration and may optionally be substituted with homocysteine (D or L).
• the Tyr residues may be substituted with the 3-hydroxyl or 2-hydroxyl isomers and corresponding O-sulpho- and O- phospho-derivatives.
• the acidic amino acid residues may be substituted with any synthetic acidic bioisoteric amino acid surrogate, e.g., tetrazolyl derivatives of Gly and Ala.
• the present invention is directed to ⁇ -conotoxin peptides having the general formula III: Xaa 1 -Xaa 2 -Xaa 3 -Xaa 4 -Xaa 5 -Cys-Cys-Xaa 6 -Xaa 7 -Xaa 8 -Xaa 9 -Cys-Xaa, 0 -Xaa n -Xaa] 2 -Xaa, 3 -
• Xaa is Leu, Gin, Val, He, Gly, Met, Ala, Lys, N-methyl-Lys, N-N-dimethyl-Lys, N,N,N-trimethyl-Lys, Ser, Thr, Arg, homoarginine, ornithine, any unnatural basic amino acid, Asn, Glu, Gla, Gin, Phe, Trp (D or L), neo-Trp, halo-Trp or any unnatural aromatic amino acid; Xaa, 2 is Leu, Gin, Val, He, Gly, Met, Ala, Lys, N-methyl-Lys, N-N-dimethyl-Lys, N,N,N-trimethyl-Lys, Ser, Thr, Arg, homoarginine, ornithine, any unnatural basic amino acid, Asn, Glu, Gla, Gin, Phe, Trp (D or L), neo-Trp, halo-Trp or any unnatural
• Glu Gla, Gin, Asn, Asp, Pro, hydroxy-Pro, Ser, Gly, Thr, Lys, N-methyl-Lys, N-N-dimethyl-Lys, N,N,N-trimethyl-Lys, Arg, homoarginine, ornithine, any unnatural basic amino acid, Phe, His, halo- His, any unnatural aromatic amino acid, Leu, Met, Gly, Ala, Tyr, nor-Tyr, mono-halo-Tyr, di-halo- Tyr, O-sulpho-Tyr, O-phospho-Tyr, nitro-Tyr or any unnatural hydroxy containing amino acid;
• Xaa, 3 is His, halo-His, Asn, Thr, Ser, He, Val, Leu, Phe, any unnatural aromatic amino acid, Arg, homoarginine, ornithine, Lys, N-methyl-Lys, N,N-dimethyl-Lys, N,
• Xaa, 4 is Ser, Thr, Ala, Gin, Pro, hydroxy-Pro, Gly, He, Leu, Arg, ornithine, homoarginine, Lys, N-methyl-Lys, N-N-dimethyl-Lys, N,N,N-trimethyl-Lys or any unnatural basic amino acid
• Xaa, 5 is Asn, Glu, Gla, Asp, Gly, His, halo- His, Ala, Leu, Gin, Arg, ornithine, homoarginine, Lys, N-methyl-Lys, N-N-dimethyl-Lys, N,N,N- trimethyl-Lys, any unnatural basic amino acid, Tyr, nor-Tyr, mono-halo-Tyr, di-halo-Tyr, O-sulpho-
• Xaa, 8 is des-Xaa, 8 , Gly, Glu, Gla, Gin, Trp (D or L), neo, halo-Trp, any unnatural aromatic amino acid, Arg, ornithine, homoarginine, Lys, N-methyl-Lys, N-N-dimethyl-Lys, N,N,N-trimethyl-Lys or any unnatural basic amino acid;
• Xaa, 9 is des-Xaa, 9 , Ser, Thr, Val, He, Ala, Arg, ornithine, homoarginine, Lys, N-methyl-
• Xaa 20 is des-Xaa 20 .
• Xaa 2 is des-Xaa 2 ,, Asn, Pro or hydroxy- Pro
• Xaa 22 is des-Xaa 22 , Arg, ornithine, homoarginine, Lys, N-methyl-Lys, N-N-dimethyl-Lys, N,N,N-trimethyl-Lys or any unnatural basic amino acid
• Xaa 23 is des-Xaa 23 , Ser or Thr
• Xaa 24 is des- Xaa 24 , Leu,
• the C-terminus may contain a free carboxyl group or an amide group.
• the halo is preferably bromine, chlorine or iodine, more preferably iodine for His and Tyr and bromine for Trp.
• the Cys residues may be in D or L configuration and may optionally be substituted with homocysteine (D or L).
• the Tyr residues may be substituted with the 3-hydroxyl or 2-hydroxyl isomers and corresponding O-sulpho- and O-phospho-derivatives.
• the acidic amino acid residues may be substituted with any synthetic acidic bioisoteric amino acid surrogate, e.g., tetrazolyl derivatives of Gly and Ala.
• the present invention is also directed to novel specific ⁇ -conotoxin peptides of general formula I having the formulas:
• the halo is preferably bromine, chlorine or iodine, more preferably iodine for Tyr and bromine for Trp.
• the His residues may be substituted with halo-His;
• the Arg residues may be substituted by Lys, ornithine, homoargine, N- methyl-Lys, N,N-dimethyl-Lys, N,N,N-trimethyl-Lys or any unnatural basic amino acid;
• the Lys residues may be substituted by Arg, ornithine, homoargine, N-methyl-Lys, N-N-dimethyl-Lys, N,N,N-trimethyl-Lys or any unnatural basic amino acid;
• the Tyr residues may be substituted with any unnatural hydroxy containing amino acid;
• the Ser residues may be substituted with Thr;
• the Cys residues may be in D or L configuration and may optionally be substituted with homocysteine (D or L).
• the Tyr residues may be substituted with the 3-hydroxyl or 2-hydroxyl isomers and corresponding O-sulpho- and O-phospho-derivatives.
• the acidic amino acid residues may be substituted with any synthetic acidic bioisoteric amino acid surrogate, e.g., tetrazolyl derivatives of Gly and Ala. More specifically, the present invention is directed to the following ⁇ -conotoxin peptides of general formula I:
• Iml J SEQ ID NO:4, wherein Xaa, is Glu and Xaa 2 is Lys;
• Iml .2 SEQ ID NO:5, wherein Xaa 3 is Trp; Rgl .2: SEQ ID NO.6;
• Rgl .6 SEQ ID NO:7, wherein Xaa 4 is Tyr;
• Rgl .6A SEQ ID NO:8, wherein Xaa 4 is Tyr and Xaa 5 is Pro;
• Rgl .7 SEQ ID NO:9, wherein Xaa 4 is Tyr and Xaa 5 is Pro;
• Rgl .9 SEQ ID NOJ 0, wherein Xaa 3 is Trp and Xaa 5 is Pro;
• Rgl J 0 SEQ ID NOJ 1 , wherein Xaa, is Glu, Xaa 2 is Lys, Xaa 3 is Trp and Xaa 5 is
• Rgl.l 1 SEQ ID NOJ2, wherein Xaa 2 is Lys and Xaa 5 is Pro.
• the C-terminus of Iml J, Rgl.7 an Rgl JO preferably contains a free carboxyl group.
• the C- terminus of Iml.2, Rgl.2, Rgl.6, Rgl.6A, Rgl.9 and Rgl.l 1 preferably contains an amide group.
• the present invention is further directed to novel specific ⁇ -conotoxin peptides of general formula II having the formulas:
• Cys-Cys-Ser-Asp-Xaa 5 -Ala-Cys-Xaa 2 -Gln-Thr-Xaa 5 -Gly-Cys-Arg (SEQ ID NO: 13); Cys-Cys-Xaa,-Asn-Xaa 5 -Ala-Cys-Arg-His-Thr-Gln-Gly-Cys (SEQ ID NO: 14); Gly-Cys-Cys-Xaa 3 -His-Xaa 5 -Ala-Cys-Gly-Arg-His-Xaa 4 -Cys (SEQ ID NO: 15); Ala-Xaa 5 -Cys-Cys-Asn-Asn-Xaa 5 -Ala-Cys-Val-Xaa 2 -His-Arg-Cys (SEQ ID NO: 16);
• Gly-Cys-Cys-Gly-Asn-Xaa 5 -Asp-Cys-Thr-Ser-His-Ser-Cys (SEQ ID NO:21); Gly-Cys-Cys-Ser-Asn-Xaa 5 -Xaa 5 -Cys-Ala-His-Asn-Asn-Xaa 5 -Asp-Cys-Arg (SEQ ID NO:42);
• Cys-Cys-Ser-Asn-Xaa 5 -Thr-Cys-Xaa 2 -Xaa,-Thr-Xaa 4 -Gly-Cys (SEQ ID NO: 158); Cys-Cys-Ala-Asn-Xaa 5 -Ile-Cys-Xaa 2 -Asn-Thr-Xaa Gly-Cys (SEQ ID NO: 159);
• Asp-Cys-Cys-Ser-Asn-Xaa 5 -Xaa 5 -Cys-Ala-His-Asn-Asn-Xaa 5 -Asp-Cys-Arg (SEQ ID NO: 169), wherein Xaa, is Glu or ⁇ -carboxy-Glu (Gla); Xaa 2 is Lys, N-methyl-Lys, N-N-dimethyl-Lys or N,N,N-trimethyl-Lys; Xaa 3 is Trp (D or L), halo-Trp or neo-Trp; Xaa 4 is Tyr, nor-Tyr, mono-halo- Tyr, di-halo-Tyr, O-sulpho-Tyr, O-phospho-Tyr or nitro-Tyr; and Xaa 5 is Pro or hydroxy-Pro; and the C-terminus contains a carboxyl or amide group.
• the halo is preferably bromine, chlorine or iodine, more preferably iodine for Tyr and bromine for Trp.
• the His residues may be substituted with halo-His;
• the Arg residues may be substituted by Lys, ornithine, homoargine, N- methyl-Lys, N,N-dimethyl-Lys, N,N,N-trimethyl-Lys or any unnatural basic amino acid;
• the Lys residues may be substituted by Arg, ornithine, homoargine, N-methyl-Lys, N,N-dimethyl-Lys, N,N,N-trimethyl-Lys or any unnatural basic amino acid;
• the Tyr residues may be substituted with any unnatural hydroxy containing amino acid;
• the Ser residues may be substituted with Thr;
• the Cys residues may be in D or L configuration and may optionally be substituted with homocysteine (D or L).
• the Tyr residues may be substituted with the 3-hydroxyl or 2-hydroxyl isomers and corresponding O-sulpho- and O-phospho-derivatives.
• the acidic amino acid residues may be substituted with any synthetic acidic bioisoteric amino acid surrogate, e.g., tetrazolyl derivatives of Gly and Ala. More specifically, the present invention is directed to the following ⁇ -conotoxin peptides of general formula II:
• Snl .1 SEQ ID NO: 13, wherein Xaa 2 is Lys and Xaa 5 is Pro;
• Snl .2 SEQ ID NOJ4, wherein Xaa, is Glu and Xaa 5 is Pro;
• SI 1.3 SEQ ID NO: 15, wherein Xaa 3 is Trp, Xaa 4 is Tyr and Xaa 5 is Pro;
• A1.2 SEQ ID NOJ6, wherein Xaa 2 is Lys and Xaa 5 is Pro;
• Bui J SEQ ID NOJ 7, wherein Xaa 2 is Lys and Xaa 5 is Pro;
• Bui .2 SEQ ID NO: 18, wherein Xaa 2 is Lys and Xaa 5 is Pro;
• Bui .3 SEQ ID NO: 19, wherein Xaa, is Glu, Xaa 3 is Trp and Xaa 5 is Pro;
• Bui .4 SEQ ID NO:20, wherein Xaa 4 is Tyr and Xaa 5 is Pro ;
• Crl.3 SEQ ID NO.21, wherein Xaa 5 is Pro;
• Msl .7 SEQ ID NO: 154, wherein Xaa 2 is Lys, Xaa 3 is Trp, Xaa 4 is Tyr and Xaa 5 is
• Msl.2 SEQ ID NO: 156, wherein Xaa 5 is Pro;
• Msl .3 SEQ ID NO: 157, wherein Xaa 2 is Lys, Xaa 3 is Trp, Xaa 4 is Tyr and Xaa 5 is
• Msl .4 SEQ ID NOJ58, wherein Xaa, is Glu, Xaa 2 is Lys, Xaa 4 is Tyr and Xaa 5 is Pro;
• Ms 1.5 SEQ ID NO: 159, wherein Xaa 2 is Lys and Xaa 5 is Pro;
• Msl .8 SEQ ID NO: 160, wherein Xaa, is Glu, Xaa 2 is Lys, Xaa 4 is Tyr and Xaa 5 is
• Msl .9 SEQ ID NOJ61, wherein Xaa, is Glu, Xaa 2 is Lys, Xaa 4 is Tyr and Xaa 5 is Pro;
• Btl.7 SEQ ID NOJ62, wherein Xaa 2 is Lys, Xaa 4 is Tyr and Xaa 5 is Pro;
• Lvl .5 SEQ ID NO: 163, wherein Xaa 5 is Pro;
• Msl J 0 SEQ ID NO: 164, wherein Xaa 2 is Lys, Xaa 4 is Tyr and Xaa 5 is Pro;
• Oml J SEQ ID NO: 165, wherein Xaa 4 is Tyr and Xaa 5 is Pro;
• R1.6 SEQ ID NO: 166, wherein Xaa 4 is Tyr and Xaa 5 is Pro;
• R1.7 SEQ ID NO: 167, wherein Xaa 4 is Tyr and Xaa 5 is Pro; Vrl J : SEQ ID NO: 168, wherein Xaa 5 is Pro; and
• Vrl .2 SEQ ID NO: 169, wherein Xaa 5 is Pro.
• the C-terminus preferably contains a carboxyl group for the peptides SnlJ, Snl.2, Crl.3, DilJ, Msl .2, Msl .4, Msl .5, Msl.8, Msl.9, Vrl .l and Vrl .2.
• the C-terminus of the other peptides preferably contains an amide group.
• the present invention is also directed to novel specific ⁇ -conotoxin peptides of general formula III having the formulas:
• Xaa 5 -Gln-Cys-Cys-Ser-His-Xaa 5 -Ala-Cys-AsnNal-Asp-His-Xaa 5 -Gly- ⁇ e-Cys-Arg SEQ ID ⁇ O.28
• Xaa 5 -Gln-Cys-Cys-Ser-His-Xaa 5 -Ala-Cys-Asn-Val-Asp-His-Xaa 5 -Xaa,-Thr-Cys-Arg SEQ ID ⁇ O.28
• Gly-Gly-Cys-Cys-Ser-Asn-Xaa 5 -Ala-Cys-Leu-Val-Asn-His-Leu-Xaa,-Met-Cys SEQ ID NO:215
• Arg-Asp-Xaa 5 -Cys-Cys-Phe-Asn-Xaa 5 -Ala-Cys-Asn-Val-Asn-Asn-Xaa 5 -Gln-Ile-Cys SEQ IDNO:216
• Xaa is Glu or ⁇ -carboxy-Glu (Gla);
• Xaa 2 is Lys, N-methyl-Lys, N-N-dimethyl-Lys or N,N,N-trimethyl-Lys;
• Xaa 3 is Trp (D or L), halo-Trp or neo-Trp;
• Xaa 4 is Tyr, nor-Tyr, mono-halo- Tyr, di-halo-Tyr, O-sulpho-Tyr, O-phospho-Tyr or nitro-Tyr; and
• Xaa 5 is Pro or hydroxy-Pro;
• Xaa 6 is Gin or
• the halo is preferably bromine, chlorine or iodine, more preferably iodine for Tyr and bromine for Trp.
• the His residues may be substituted with halo-His;
• the Arg residues may be substituted by Lys, ornithine, homoargine, N-methyl-Lys, N-N-dimethyl-Lys, N,N,N-trimethyl-Lys or any unnatural basic amino acid;
• the Lys residues may be substituted by Arg, ornithine, homoargine, N-methyl-Lys, N,N-dimethyl-Lys, N,N,N-trimethyl-Lys or any unnatural basic amino acid;
• the Tyr residues may be substituted with any unnatural hydroxy containing amino acid;
• the Ser residues may be substituted with Thr;
• the Cys residues may be in D or L configuration and may optionally be substituted with homocysteine (D or L).
• the Tyr residues may be substituted with the 3-hydroxyl or 2-hydroxyl isomers and corresponding O-sulpho- and O- phospho-derivatives.
• the acidic amino acid residues may be substituted with any synthetic acidic bioisoteric amino acid surrogate, e.g., tetrazolyl derivatives of Gly and Ala.
• the present invention is directed to the following ⁇ -conotoxin peptides of general formula III:
• Sml SEQ ID NO:22, wherein Xaa, s Glu and Xaa 5 is Pro;
• OB-29 SEQ ID NO:23, wherein Xaa, s Glu, Xaa 3 is Tyr and Xaa 5 is Pro;
• TxlJ SEQ ID NO:24, wherein Xaa, s Glu and Xaa 5 is Pro;
• R1JA SEQ ID NO:25, wherein Xaa, s Glu and Xaa 5 is Pro;
• R1JB SEQ ID NO:26, wherein Xaa, s Glu and Xaa 5 is Pro;
• Om-9 SEQ ID NO:27, wherein Xaa, s Glu and Xaa 5 is Pro;
• Btl.2 SEQ ID NO:33, wherein Xaa, s Glu and Xaa 5 is Pro;
• Btl.4 SEQ ID NO:34, wherein Xaa, s Glu and Xaa 5 is Pro;
• Dal.l SEQ ID NO:35, wherein Xaa, s Glu and Xaa 5 is Pro;
• OB-20 SEQ ID NO:36, wherein Xaa, is Glu, Xaa 2 is Lys and Xaa 5 is Pro;
• TI SEQ ID NO:37, wherein Xaa, is Glu and Xaa 5 is Pro;
• TIB SEQ ID NO:38, wherein Xaa, is Glu and Xaa 5 is Pro;
• Pnl J SEQ ID NO:39, wherein Xaa 5 is Pro;
• Pnl.2 SEQ ID NO:40, wherein Xaa, is Glu and Xaa 5 is Pro;
• Tl SEQ ID NO.41 , wherein Xaa 2 is Lys and Xaa 5 is Pro;
• TIA SEQ ID NO:43, wherein Xaa 5 is Pro;
• Dal .2 SEQ ID NO:44, wherein Xaa 5 is Pro;
• Crl .2 SEQ ID NO:45, wherein Xaa, is Glu and Xaa 5 is Pro;
• Sll .2 SEQ ID NO:46, wherein Xaa, is Glu, Xaa 2 is Lys and Xaa 5 is Pro;
• Txl .3 SEQ ID NO:47, wherein Xaa, is Glu and Xaa 5 is Pro;
• Txl .2 SEQ ID NO:50, wherein Xaa 5 is Pro;
• Om-35 SEQ ID NO:51, wherein Xaa, is Glu and Xaa 5 is Pro;
• Sll J SEQ ID NO:52, wherein Xaa, is Glu, Xaa 3 is Trp, Xaa 4 is Tyr and Xaa 5 is
• S SI111..77 SEQ ID NO:54, wherein Xaa, is Glu Xaa 4 is Tyr and Xaa 5 is Pro;
• Btl .l SEQ ID NO:55, wherein Xaa, is Glu Xaa 4 is Tyr and Xaa 5 is Pro;
• Bt 1.3: SEQ ID NO:56, wherein Xaa, is Glu Xaa 4 is Tyr and Xaa 5 is Pro;
• Btl .5 SEQ ID NO:57, wherein Xaa, is Glu Xaa 4 is Tyr and Xaa 5 is Pro;
• A1.4 SEQ ID NO: 170, wherein Xaa, is Glu and Xaa 5 is Pro;
• Afl .l SEQ ID NO: 173, wherein Xaa, is Glu Xaa 4 is Tyr, Xaa 5 is Pro and Xaa 6 is
• Afl .2 SEQ ID NO: 174, wherein Xaa, is Glu and Xaa 5 is Pro;
• Arl.5 SEQ ID NO: 178, wherein Xaa, is Glu and Xaa 5 is Pro;
• Arl .6 SEQ ID NO: 179, wherein Xaa, is Glu, Xaa 2 is Lys, Xaa 3 is T ⁇ , Xaa 4 is Try and Xaa 5 is Pro;
• Ayl .2 SEQ ID NOJ 80, wherein Xaa 4 is Tyr and Xaa 5 is Pro;
• Ay 1.3 SEQ ID NO: 181, wherein Xaa 5 is Pro;
• Bnl A SEQ ID NO: 182, wherein Xaa 4 is Tyr and Xaa 5 is Pro;
• Btl .8 SEQ ID NOJ 83, wherein Xaa, is Glu and Xaa 5 is Pro
• Btl .9 SEQ ID NO: 184, wherein Xaa, is Glu, Xaa 4 is Tyr and Xaa 5 is Pro;
• Xaa 5 is Pro and Xaaa is Gin;
• C1.2 SEQ ID NO:187, wherein Xaa, is Glu and Xaaj is Pro;
• Epl .2 SEQ ID NOJ 89, wherein Xaa, is Glu, Xaa 2 is Lys, Xaa 3 is T ⁇ and Xaa 5 is
• Gl .l SEQ ID NO: 190, wherein Xaa 5 is Pro; G1.3: SEQ ID NO-191, wherein Xaa 5 is Pro;
• Iml .3 SEQ ID NO: 192, wherein Xaa 5 is Pro;
• Lvl.2 SEQ ID NO: 193, wherein Xaa, is Glu and Xaa 5 is Pro;
• Lvl .3 SEQ ID NO: 194, wherein Xaa 2 is Lys and Xaa 5 is Pro;
• Lvl A SEQ ID NO: 195, wherein Xaa, is Glu and Xaa 5 is Pro
• Lvl .6 SEQ ID NO: 196, wherein Xaa 5 is Pro
• Lvl .7 SEQ ID NO: 197, wherein Xaa, is Glu and Xaa 5 is Pro;
• Lvl .8 SEQ ID NO: 198, wherein Xaa 5 is Pro;
• Lvl .9 SEQ ID NO: 199, wherein Xaa 4 is Tyr and Xaa 5 is Pro;
• Lvl JO SEQ ID NO:200, wherein Xaa, is Glu; Mr 1.3: SEQ ID NO-201, wherein Xaa 4 is Tyr and Xaa 5 is Pro;
• Mrl .4 SEQ ID NO:202, wherein Xaa 5 is Pro;
• Msl J SEQ ID NO:203, wherein Xaa 2 is Lys, Xaa 4 is Tyr and Xaa 5 is Pro; Msl .6: SEQ ID NO:204, wherein Xaa 4 is Tyr and Xaa 5 is Pro;
• O1 J SEQ ID NO:205, wherein Xaa 2 is Lys, Xaa 4 is Tyr and Xaa 5 is Pro;
• P1.4 SEQ ID NO:215, wherein Xaa, is Glu and Xaa 5 is Pro;
• P1.8 SEQ ID NO:218, wherein Xaa 5 is Pro;
• the C-terminus of the other peptides preferably contains an amide group.
• the present invention is also directed to the novel specific ⁇ -contoxin peptides having the formulas: Cys-Cys-Thr-Ile-Xaa 5 -Ser-Cys-Xaa 4 -Xaa,-Xaa 2 -Xaa 2 -Xaa 2 -He-Xaa 2 -Ala-Cys-Val-Phe(SEQ ID NO:
• halo is preferably bromine, chlorine or iodine, more preferably iodine for Tyr.
• the His residues may be substituted with halo-His;
• the Arg residues may be substituted by Lys, ornithine, homoargine, N-methyl-Lys, N-N-dimethyl-Lys, N,N,N-trimethyl-Lys or any unnatural basic amino acid;
• the Lys residues may be substituted by Arg, ornithine, homoargine, N- methyl-Lys, N-N-dimethyl-Lys, N,N,N-trimethyl-Lys or any unnatural basic amino acid;
• the Tyr residues may be substituted with any unnatural hydroxy containing amino acid;
• the Ser residues may be substituted with Thr;
• the Thr residues may be substituted with Ser; and
• the Phe residues may be substituted with any unnatural aromatic amino acid.
• the Cys residues may be in D or L configuration and may optionally be substituted with homocysteine (D or L).
• the Tyr residues may be substituted with the 3-hydroxyl or 2-hydroxyl isomers and corresponding O-sulpho- and O- phospho-derivatives.
• the acidic amino acid residues may be substituted with any synthetic acidic bioisoteric amino acid surrogate, e.g., tetrazolyl derivatives of Gly and Ala.
• the present invention is directed to the following ⁇ -conotoxin peptides: Gl .2: SEQ ID NO:231 , wherein Xaa, is Glu, Xaa 2 is Lys, Xaa 4 is Tyr and Xaa 5 is
• RglJ2 SEQ ID NO:232, wherein Xaa 2 is Lys and Xaa 5 is Pro.
• the C-terminus of G1.2 preferably contains a carboxyl group, and the C-terminus of RglJ2 preferably contains an amide group.
• unnatural aromatic amino acid include, but are not limited to, such as nitro-Phe,
• 4-substituted-Phe wherein the substituent is C,-C 3 alkyl, carboxyl, hyrdroxymethyl, sulphomethyl, halo, phenyl, -CHO, -CN, -SO 3 H and -NHAc.
• unnatural hydroxy containing amino acid include, but are not limited to, such as 4-hydroxymethyl-Phe, 4-hydroxyphenyl-Gly, 2,6- dimethyl-Tyr and 5-amino-Tyr.
• unnatural basic amino acids include, but are not limited to, N- 1 -(2 -pyrazolinyl)-Arg, 2-(4-piperinyl)-Gly, 2-(4-piperinyl)-Ala, 2-[3-(2S)pyrrolininyl)- Gly and 2-[3-(2S)pyrrolininyl)-Ala.
• unnatural basic amino acids unnatural hydroxy containing amino acids or unnatural aromatic amino acids are described in Building Block
• the Asn residues may be modified to contain an N-glycan and the Ser and Thr residues may be modified to contain an O-glycan.
• a glycan shall mean any N-, S- or O-linked mono-, di-, tri-, poly- or oligosaccharide that can be attached to any hydroxy, amino or thiol group of natural or modified amino acids by synthetic or enzymatic methodologies known in the art.
• the monosaccharides making up the glycan can include
• D-allose D-altrose, D-glucose, D-mannose, D-gulose, D-idose, D-galactose, D-talose, D- galactosamine, D-glucosamine, D-N-acetyl-glucosamine (GlcNAc), D-N-acetyl-galactosamine (GalNAc), D-fucose or D-arabinose.
• These saccharides may be structurally modified, e.g., with one or more O-sulfate, O-phosphate, O-acetyl or acidic groups, such as sialic acid, including combinations thereof.
• the gylcan may also include similar polyhydroxy groups, such as D- penicillamine 2,5 and halogenated derivatives thereof or polypropylene glycol derivatives.
• the glycosidic linkage is beta and 1-4 or 1-3, preferably 1-3.
• the linkage between the glycan and the amino acid may be alpha or beta, preferably alpha and is 1-.
• Core O-glycans have been described by Van de Steen et al. (1998), inco ⁇ orated herein by reference.
• Mucin type O-linked oligosaccharides are attached to Ser or Thr (or other hydroxylated residues of the present peptides) by a GalNAc residue.
• the monosaccharide building blocks and the linkage attached to this first GalNAc residue define the "core glycans,” of which eight have been identified.
• the type of glycosidic linkage (orientation and connectivities) are defined for each core glycan. Suitable glycans and glycan analogs are described further in U.S. Serial No. 09/420,797, filed 19 October 1999 and in PCT Application No.
• a preferred glycan is Gal( ⁇ l ⁇ 3)GalNAc( ⁇ l-).
• pairs of Cys residues may be replaced pairwise with Ser/(Glu or Asp) or Lys/(Glu or Asp) combinations. Sequential coupling by known methods (Barnay et al., 2000; Hruby et al., 1994; Bitan et al., 1997) allows replacement of native Cys bridges with lactam bridges.
• the present invention is further directed to propeptides and nucleic acid sequences encoding the propeptides or peptides as described in further detail herein.
• the invention relates to relatively short peptides (termed ⁇ -conotoxins herein), about 10-30 residues in length, which are naturally available in minute amounts in the venom of the cone snails or analogous to the naturally available peptides, and which preferably include two disulfide bonds.
• the present invention in another aspect, relates to a pharmaceutical composition
• a pharmaceutical composition comprising an effective amount of an ⁇ -conotoxin peptide.
• Such a pharmaceutical composition has the capability of acting as antagonists for nicotinic acetylcholine receptors.
• the ⁇ - conotoxins with specificity for neuromuscular junction nicotinic acetylcholine receptors are used as neuromuscular blocking agents for use in conjunction with surgery, as disclosed in U.S. patent application Serial No. 09/ , filed 21 January 2000 (Attorney Docket No. 2314-178.A) and international patent application No. PCT/US00/ , filed 21 January 2000 (Attorney Docket No.
• ⁇ -conotoxins with specificity for neuronal nicotinic acetylcholine receptors are used for treating disorders regulated at neuronal nicotinic acetylcholine receptors.
• disorders include, but are not limited to, cardiovascular disorders, gastric motility disorders, urinary incontinence, nicotine addiction, mood disorders (such as bipolar disorder, unipolar depression, dysthymia and seasonal effective disorder) and small cell lung carcinoma, as well as the localization of small cell lung carcinoma.
• ⁇ -conotoxin peptides described herein are sufficiently small to be chemically synthesized.
• General chemical syntheses for preparing the foregoing ⁇ -conotoxin peptides are described hereinafter.
• Various ones of the ⁇ -conotoxin peptides can also be obtained by isolation and purification from specific Conus species using the technique described in U.S. Patent No. 4,447,356 (Olivera et al., 1984), the disclosure of which is incorporated herein by reference.
• the ⁇ -conotoxin peptides of the present invention can be obtained by purification from cone snails, because the amounts of ⁇ -conotoxin peptides obtainable from individual snails are very small, the desired substantially pure ⁇ -conotoxin peptides are best practically obtained in commercially valuable amounts by chemical synthesis using solid-phase strategy.
• the yield from a single cone snail may be about 10 micrograms or less of ⁇ -conotoxin peptide.
• substantially pure is meant that the peptide is present in the substantial absence of other biological molecules of the same type; it is preferably present in an amount of at least about 85% purity and preferably at least about 95% purity. Chemical synthesis of biologically active ⁇ -conotoxin peptides depends of course upon correct determination of the amino acid sequence.
• the ⁇ -conotoxin peptides can also be produced by recombinant DNA techniques well known in the art. Such techniques are described by Sambrook et al. (1989). The peptides produced in this manner are isolated, reduced if necessary, and oxidized to form the correct disulfide bonds.
• One method of forming disulfide bonds in the conantokin peptides of the present invention is the air oxidation of the linear peptides for prolonged periods under cold room temperatures or at room temperature. This procedure results in the creation of a substantial amount of the bioactive, disulfide-linked peptides.
• the oxidized peptides are fractionated using reverse-phase high performance liquid chromatography (HPLC) or the like, to separate peptides having different linked configurations. Thereafter, either by comparing these fractions with the elution of the native material or by using a simple assay, the particular fraction having the correct linkage for maximum biological potency is easily determined. However, because of the dilution resulting from the presence of other fractions of less biopotency, a somewhat higher dosage may be required.
• HPLC reverse-phase high performance liquid chromatography
• the peptides are synthesized by a suitable method, such as by exclusively solid-phase techniques, by partial solid-phase techniques, by fragment condensation or by classical solution couplings.
• the peptide chain can be prepared by a series of coupling reactions in which constituent amino acids are added to the growing peptide chain in the desired sequence.
• various coupling reagents e.g., dicyclohexylcarbodiimide or diisopropylcarbonyldimidazole
• various active esters e.g., esters of N-hydroxyphthalimide or N- hydroxy-succinimide
• the various cleavage reagents to carry out reaction in solution, with subsequent isolation and purification of intermediates, is well known classical peptide methodology.
• the protecting group preferably retains its protecting properties and is not split off under coupling conditions
• the protecting group should be stable under the reaction conditions selected for removing the ⁇ -amino protecting group at each step of the synthesis
• the side chain protecting group must be removable, upon the completion of the synthesis containing the desired amino acid sequence, under reaction conditions that will not undesirably alter the peptide chain.
• peptides are not so prepared, they are preferably prepared using the Merrifield solid-phase synthesis, although other equivalent chemical syntheses known in the art can also be used as previously mentioned. Solid-phase synthesis is commenced from the C-terminus of the peptide by coupling a protected ⁇ -amino acid to a suitable resin.
• Such a starting material can be prepared by attaching an ⁇ -amino-protected amino acid by an ester linkage to a chloromethylated resin or a hydroxymethyl resin, or by an amide bond to a benzhydrylamine (BHA) resin or para- methylbenzhydrylamine (MBHA) resin.
• BHA benzhydrylamine
• MBHA para- methylbenzhydrylamine
• Preparation of the hydroxymethyl resin is described by Bodansky et al. (1966). Chloromethylated resins are commercially available from Bio Rad Laboratories (Richmond, CA) and from Lab. Systems, Inc. The preparation of such a resin is described by Stewart and Young (1969).
• BHA and MBHA resin supports are commercially available, and are generally used when the desired polypeptide being synthesized has an unsubstituted amide at the C-terminus.
• solid resin supports may be any of those known in the art, such as one having the formulae -O-CH 2 -resin support, -NH BHA resin support, or -NH-MBHA resin support.
• unsubstituted amide use of a BHA or MBHA resin is preferred, because cleavage directly gives the amide.
• N-methyl amide is desired, it can be generated from an N-methyl BHA resin. Should other substituted amides be desired, the teaching of U.S. Patent No.
• the C-terminal amino acid protected by Boc or Fmoc and by a side-chain protecting group, if appropriate, can be first coupled to a chloromethylated resin according to the procedure set forth in K. Horiki et al. (1978), using KF in DMF at about 60°C for 24 hours with stirring, when a peptide having free acid at the C-terminus is to be synthesized.
• the ⁇ -amino protecting group is removed, as by using trifluoroacetic acid (TFA) in methylene chloride or TFA alone.
• TFA trifluoroacetic acid
• N-N'-dicyclohexylcarbodiimide DCC, DIC, HBTU, HATU, TBTU in the presence of HoBt or HoAt.
• the activating reagents used in the solid phase synthesis of the peptides are well known in the peptide art.
• suitable activating reagents are carbodiimides, such as N,N'- diisopropylcarbodiimide and N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide.
• Other activating reagents and their use in peptide coupling are described by Schroder & Lubke (1965) and Kapoor (1970).
• Each protected amino acid or amino acid sequence is introduced into the solid-phase reactor in about a twofold or more excess, and the coupling may be carried out in a medium of dimethylformamide (DMF):CH 2 C1 2 (1 J) or in DMF or CH 2 C1 2 alone.
• DMF dimethylformamide
• the coupling procedure is repeated before removal of the ⁇ -amino protecting group prior to the coupling of the next amino acid.
• the success of the coupling reaction at each stage of the synthesis if performed manually, is preferably monitored by the ninhydrin reaction, as described by Kaiser et al. (1970).
• Coupling reactions can be performed automatically, as on a Beckman 990 automatic synthesizer, using a program such as that reported in Rivier et al. (1978).
• the intermediate peptide can be removed from the resin support by treatment with a reagent, such as liquid hydrogen fluoride or
• the Boc protecting group is preferably first removed using trifluoroacetic acid (TFA)/ethanedithiol prior to cleaving the peptide from the resin with HF to eliminate potential S- alkylation.
• TFA trifluoroacetic acid
• one or more scavengers such as anisole, cresol, dimethyl sulfide and methylethyl sulfide are included in the reaction vessel.
• Cyclization of the linear peptide is preferably affected, as opposed to cyclizing the peptide while a part of the peptido-resin, to create bonds between Cys residues.
• fully protected peptide can be cleaved from a hydroxymethylated resin or a chloromethylated resin support by ammonolysis, as is well known in the art, to yield the fully protected amide intermediate, which is thereafter suitably cyclized and deprotected.
• deprotection, as well as cleavage of the peptide from the above resins or a benzhydrylamine (BHA) resin or a methylbenzhydrylamine (MBHA), can take place at 0°C with hydrofluoric acid (HF) or TFA, followed by oxidation as described above.
• the peptides are also synthesized using an automatic synthesizer.
• Amino acids are sequentially coupled to an MBHA Rink resin (typically 100 mg of resin) beginning at the C- terminus using an Advanced Chemtech 357 Automatic Peptide Synthesizer. Couplings are carried out using 1,3-diisopropylcarbodimide in N-methylpyrrolidinone (NMP) or by 2-(lH-benzotriazole- l-yl)-lJ,3,3-tetramethyluronium hexafluorophosphate (HBTU) and diethylisopro- pylethylamine (DIEA).
• NMP N-methylpyrrolidinone
• HBTU 2-(lH-benzotriazole- l-yl)-lJ,3,3-tetramethyluronium hexafluorophosphate
• DIEA diethylisopro- pylethylamine
• compositions containing a compound of the present invention or its pharmaceutically acceptable salts as the active ingredient can be prepared according to conventional pharmaceutical compounding techniques. See, for example, Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, PA). Typically, an antagonistic amount of the active ingredient will be admixed with a pharmaceutically acceptable carrier.
• the carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g., intravenous, oral or parenteral.
• the compositions may further contain antioxidizing agents, stabilizing agents, preservatives and the like.
• the compounds can be formulated into solid or liquid preparations such as capsules, pills, tablets, lozenges, melts, powders, suspensions or emulsions.
• any of the usual pharmaceutical media may be employed, such as, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, suspending agents, and the like in the case of oral liquid preparations (such as, for example, suspensions, elixirs and solutions); or carriers such as starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in the case of oral solid preparations (such as, for example, powders, capsules and tablets). Because of their ease in administration, tablets and capsules represent the most advantageous oral dosage unit form, in which case solid pharmaceutical carriers are obviously employed.
• tablets may be sugar-coated or enteric-coated by standard techniques.
• the active agent can be encapsulated to make it stable to passage through the gastrointestinal tract while at the same time allowing for passage across the blood brain barrier. See for example, WO 96/11698.
• the compound may be dissolved in a pharmaceutical carrier and administered as either a solution or a suspension.
• suitable carriers are water, saline, dextrose solutions, fructose solutions, ethanol, or oils of animal, vegetative or synthetic origin.
• the carrier may also contain other ingredients, for example, preservatives, suspending agents, solubilizing agents, buffers and the like.
• the compounds When the compounds are being administered intrathecally, they may also be dissolved in cerebrospinal fluid.
• the active agent is preferably administered in an therapeutically effective amount.
• the actual amount administered, and the rate and time-course of administration, will depend on the nature and severity of the condition being treated. Prescription of treatment, e.g. decisions on dosage, timing, etc., is within the responsibility of general practitioners or spealists, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of techniques and protocols can be found in Remington 's Parmaceutical Sciences.
• the conopeptides of the present invention exhibit their effect at a dosage range from about 0.001 mg/kg to about 250 mg/kg, preferably from about 0.05 mg/kg to about 100 mg/kg of the active ingredient, more preferably from a bout 0J mg/kg to about 75 mg/kg.
• a suitable dose can be administered in multiple sub-doses per day.
• a dose or sub-dose may contain from about 0J mg to about 500 mg of the active ingredient per unit dosage form.
• a more preferred dosage will contain from about 0.5 mg to about 100 mg of active ingredient per unit dosage form. Dosages are generally initiated at lower levels and increased until desired effects are achieved.
• targeting therapies may be used to deliver the active agent more specifically to certain types of cell, by the use of targeting systems such as antibodies or cell specific ligands. Targeting may be desirable for a variety of reasons, e.g. if the agent is unacceptably toxic, or if it would otherwise require too high a dosage, or if it would not otherwise be able to enter the target cells.
• the active agents which are peptides, can also be administered in a cell based delivery system in which a DNA sequence encoding an active agent is introduced into cells designed for implantation in the body of the patient, especially in the spinal cord region.
• a cell based delivery system in which a DNA sequence encoding an active agent is introduced into cells designed for implantation in the body of the patient, especially in the spinal cord region.
• Suitable delivery systems are described in U.S. Patent No. 5,550,050 and published PCT Application Nos. WO
• Suitable DNA sequences can be prepared synthetically for each active agent on the basis of the developed sequences and the known genetic code.
• EXAMPLES The present invention is described by reference to the following Examples, which are offered by way of illustration and are not intended to limit the invention in any manner. Standard techniques well known in the art or the techniques specifically described below were utilized.
• the effluents were monitored at 220 nm. Peaks were collected, and aliquots were assayed for activity. Activity was monitored by assessing block of ⁇ 3 ⁇ 4 nAChRs expressed in Xenopus oocytes. The amino acid sequence of the purified peptides were determined by standard methods.
• the purified peptides were reduced and alkylated prior to sequencing by automated Edman degradation on an Applied Biosystems 477A Protein Sequencer with a 120A Analyzer (DNA/Peptide Facility, University of Utah) (Martinez et al., 1995; Shon et al., 1994).
• peptides Mil, AuIA, AuIB, AuIC, MAR-1, MAR-2, TI, OB- 29, Epl, S1J, Bnl J, Bnl.2, CalJ, Cal.2, CnlJ, Cnl.2 and Sml.3 were obtained.
• Cys 3 and Cys 16 were protected as the stable Cys(S- acetamidomethyl), while Cys 2 and Cys 8 were protected as the acid-labile Cys(S-trityl). After removal of the terminal Fmoc protecting group and cleavage of the peptides from the resins, the released peptides were precipitated by filtering the reaction mixture into -10°C methyl t-butyl ether, which removed the protecting groups except on Cys 3 and Cys 16 .
• the peptides were dissolved in 0.1% TFA and 60% acetonitrile and purified by RPLC on a Vydac C, 8 preparative column (22 x 250 mm) and eluted at a flow rate of 20 mL/min with a gradient of acetonitrile in 0.1% TFA.
• the disulfide bridges in the three conopeptides were formed as described in Cartier et al.
• cDNA libraries was prepared from Conus venom duct using conventional techniques.
• DNA from single clones was amplified by conventional techniques using primers which correspond approximately to the Ml 3 universal priming site and the Ml 3 reverse universal priming site. Clones having a size of approximately 300 nucleotides were sequenced and screened for similarity in sequence to known ⁇ -conotoxins. The DNA sequences and encoded propeptide or peptide sequences are set forth in Tables 1-134.

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• 2000
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