USOO6268.473B1 (12) United States Patent (10) Patent No.: US 6,268,473 B1 Olivera et al. (45) Date of Patent: Jul. 31, 2001

(54) c-CONOTOXIN PEPTIDES Bevan (1997). J. Clin. Anesthesia 9:365,375, 385, 395. Bowman (1997). Asia Pacific J. Pharmacol. 12:57-64. (75) Inventors: Baldomero M. Olivera, Salt Lake City; Bren et al. (2000). J. Biol. Chem.275(17): 12692–12700. Richard T. Layer, Sandy; Maren Jacobsen et al. (1999). Biochem. 38:13310–13315. Watkins, Salt Lake City; David R. Hann et al. (1994). Biochem. 33:14058–14063. Hillyard, Salt Lake City; J. Michael Groebe et al. (1997). Biochem. 36:6469–6474. McIntosh, Salt Lake City, all of UT Groebe et al. (1995). Mol. Pharmacol. 48:105-111. (US); Robert Schoenfeld, Sacramento, Hann et al. (1997). Biochem. 36:9051-9056. CA (US); Robert M. Jones, Salt Lake Almquist et al. (1989). Int. J. Peptide Protein Res. City, UT (US) 34:455-462. McIntosh et al. (1982). Arch. Biochem. & BiophyS. (73) Assignees: University of Utah Research 218(1):329–334. Foundation; Cognetix, Inc., both of Richard M. Hann, et al., “The C-Conotoxins GI and MI Salt Lake City, UT (US) Distinguish between the Nicotinic Acetylcholine Receptor Agonist Sites while SI Does Not’, Biochemistry vol. 33, No. (*) Notice: Subject to any disclaimer, the term of this 47, 1994, pp. 14058–14063. patent is extended or adjusted under 35 Duncan R. Groebe, et al., “Determinants Involved in the U.S.C. 154(b) by 0 days. Affinity of C-Conotoxins GI and SI for the Muscle Subtytpe of Nicotinic Acetylcholine Receptors”, Biochemistry, vol. (21) Appl. No.: 09/488,799 36, No. 21, 1997, pp. 6469–6474. Duncan R. Groebe, et al., “O-Conotoxins Selectively Inhibit (22) Filed: Jan. 21, 2000 One of the Two Acetycholine Binding Sites of Nicotinic Related U.S. Application Data Receptors’, Molecular Pharmacology, vol. 48, 1995, pp. (60) Provisional application No. 60/116,881, filed on Jan. 22, 105-111. 1999, and provisional application No. 60/116,882, filed on Ronald G. Almquist, et al., “Paralytic activity of Jan. 22, 1999. des-Glu")conotoxin GI analogs in the mouse diaphragm", Int. J. Peptide Protein Res., vol. 34, 1989, pp. 455-462. (51) Int. Cl." ...... C07K 7/08; CO7K 14/00; Richard M. Hann, et al., “The 9-Arginine Residue of CO7K 14/435; A61K 38/10; A61K 38/17 C-Conotoxin GI is Responsible for Its Selective High Affin (52) U.S. Cl...... 530/325; 530/325; 530/324; ity for the Cy Agonist Site on the Electric Organ Acetylcho 530/328; 530/350; 435/69.1; 435/69.7; line Receptor, Biochemistry, vol. 36, No. 29, 1997, pp. 435/320.1 90.51-9056. (58) Field of Search ...... 435/69.7, 69.1, Richard B. Jacobsen, et al., “Critical Residues Influence the 435/320.1; 530/350, 325, 328 Affinity and Selectivity of C-Conotoxin MI for Nicotinic Acetylcholine Receptors”, Biochemistry, vol. 38, No. 40, (56) References Cited 1999, pp. 13310–13315. U.S. PATENT DOCUMENTS Nina Bren, et al., “Hydrophobic Pairwise Interactions Sta bilize C-Conotoxin M1 in the Muscle Acetylcholine Recep 4,447,356 5/1984 Olivera et al...... 260/112.5 tor Binding Site”, The Journal of Biological Chemistry, vol. 5,231,011 7/1993 Hillyard et al...... 435/69.7 275, No. 17, Apr. 28, 2000, pp. 12692–12700. 5,432,155 7/1995 Olivera et al...... 514/12 Letter to the Editor, “Development of New Neuromuscular 5,514,774 5/1996 Olivera et al. ... 530/324 Blocking Drugs”, Asia Pacific Journal of Pharmacology, vol. 5,969,096 10/1999 Shon et al...... 530/325 12, 1997, pp. 57–64. FOREIGN PATENT DOCUMENTS David R. Bevan, et al. “Neuromuscular Blocking Drugs: Onset and Intubation”, Journal of Clinical Anesthesia, vol. 9, O 593 450 4/1994 (EP). O 625 162 11/1994 (EP). Sep.1997, pp. 383–396. WO 91/0798O 6/1991 (WO). (List continued on next page.) WO 93/10145 5/1993 (WO). WO 93/13128 7/1993 (WO). Primary Examiner Karen Cochrane Carlson WO 99/54350 10/1999 (WO). ASSistant Examiner Patricia Robinson WO OO/15654 3/2000 (WO). (74) Attorney, Agent, or Firm-Rothwell, Figg, Ernst & OTHER PUBLICATIONS Manbeck, pc Olivera et al. (1985). Science 230:1338–1343. (57) ABSTRACT Olivera et al. (1990). Science 249:257–263. The invention relates to relatively short peptides (termed Myers et al. (1993). Chem. Rev. 93:1923–1936. C-conotoxins herein), about 10–25 residues in length, which Blount et al. (1992). Toxicon. 30(8):835–842. are naturally available in minute amounts in the Venom of Gray et al. (1981). J. Biol. Chem. 256(10):4734–4740. the cone Snails or analogous to the naturally available Hashimoto et al. (1985). Eur, J. Pharmacol. 118:355–354. peptides, and which preferably include two disulfide bonds. Marshall et al. (1990). Toxicon. 28(2):231-234. The C-conotoxins, as described herein, are useful for as McManus et al. (1985). J. Neurosci. 5(1): 110–116. neuromuscular blocking agents, Such as muscle relaxants. McManus et al. (1981). Neurosci. Letts. 24:57-62. Smythies (1981). Medical Hypotheses 7:1457–1460. 33 Claims, 5 Drawing Sheets US 6,268.473 B1 Page 2

OTHER PUBLICATIONS Alan C. Rigby, et al. “A conotoxin from Conus textile with unusual posttranslational modifications reduces presynaptic M. McIntosh, et al., “Isolation and Structure of a Peptide Ca"influx”, Proc. Natl. Acad. Sci. USA, vol. 96, May 1999, Toxin from the Marin Snal Conus magus, Archives of pp. 5758–5763. Biochemistry and Biophysics, vol. 218, No. 1, Oct. 1, 1982, Internal Scientific Report, Metabolic Stability of CGX-1079 pp. 329-334. (C-Conopeptide Gl), May 4, 2000. Alan C. Rigby, et al., “Gamma-carboxyglutamic acid-con Craig S. Walker, et al., “The T-superfamily of Conotoxins, taining conotoxins in the Venom from Conus textile', The Journal of Biological Chemistry, vol. 274, No. 43, Oct. Abstract. 22, 1999, pp. 30664-30671. Problem Solving Report Question No.-1026746.044, Neu Baldomero M. Olivera, et al., “Generating molecular diver rex-Conotoxin, Calcium Channel Patents, May 2, 2000, sity in Conus Venoms', Abstract. Tech. Spec.-John Leavitt. U.S. Patent Jul. 31, 2001 Sheet 1 of 5 US 6,268,473 B1 14 12 87.5 UG/KG ... 100 UG/KG 150 UG/KG

O D 5% ONSET 25%RECOVERY 75% RECOVERY

FIG. 1

18 16 180 UG/KG 14 217 UG/KG 12 250 UG/KG 10

D 5%ONSET 25% RECOVERY 75% RECOVERY

FIG. 2 U.S. Patent Jul. 31, 2001 Sheet 2 of 5 US 6,268,473 B1

100 90 M1 PLOT 80 ED50-80UG/KG 70 G1 PLOT

60 ED50-120 UG/KG 50 40 30 20 10

60 80 100 120 140 160 18O DOSE (UG/KG)

FIG. 3 U.S. Patent Jul. 31, 2001 Sheet 3 of 5 US 6,268,473 B1

40

35 18.75 UG/KG 30 ZZ 28,125 UG/KG 37.5 UG/KG

25 O 75 UG/KG Y 150 UG/KG

10 5 O Obs 5%. ONSET 25% RECOVERY 75% RECOVERY

10

8 125UG/KG ZZ 137.5 UG/KG O 150 UG/KG i? 6 2 e

5Her 4

2

O 5%ONSET 25% RECOVERY 75% RECOVERY FIG. 5 U.S. Patent Jul. 31, 2001 Sheet 4 of 5 US 6,268,473 B1

100

40 30 ED50-16UG/KG

20 10 O -10 5 10 15 2O 25 30 Mm DOSE (UG/mL)

FIG. 6 U.S. Patent Jul. 31, 2001 Sheet S of 5 US 6,268,473 B1

100 O O O 90 80 70 60 50 40 30 ED50-92UG/KG 20 10

-10 70 80 90 100 110 120 130 140 150 DOSE (UG/KG)

FIG. 7 US 6,268.473 B1 1 2 C-CONOTOXIN PEPTDES 4,701.460; 4,179,507; 4,923,898; 5,015,741; and 5,260,337; as well as in Goodman and Gilman's The Pharmacological CROSS-REFERENCE TO RELATED Basis of Therapeutics, Section II, especially Chapter 11, 7th APPLICATIONS Ed. (1985) and Physicians Desk Reference, 48 Ed., pp. 689, The present application is related to U.S. provisional 758, 1362 and 1648 (1994). patent applications Serial Nos. 60/116,881 and 60/116,882, Compounds having musculoskeletal relaxing properties both filed on Jan. 22, 1999 and both incorporated herein by include (1) agents acting in the central nervous System reference. which are used to relieve pain associated with muscle This invention was made with Government support under contraction (e.g., 5-chlorobenzoxazolinone available as Grant No. PO1 GM48677 awarded by the National Institute Parafon Forte DSC from McNeil Pharmaceutical), and (2) of General Medical Sciences, National Institutes of Health, agents acting in the peripheral nervous System used prima Bethesda, Md. The United States Government has certain rily to induce muscle relaxation and hence reduce muscle rights in the invention. contraction during anesthesia. The Second group of muscle relaxants is Subdivided into two groups: (i) non-depolarizing BACKGROUND OF THE INVENTION 15 agents which inhibit the activation of muscle receptors (e.g., The invention relates to relatively short peptides (termed metocurarine iodide, d-tubocurarine, tubocurarine chloride, C-conotoxins herein), about 10–25 residues in length, which pancuronium, gallamine, diallytoiferine, toxiferine, atracu are naturally available in minute amounts in the Venom of rium besylate which is available as Tracrium from the cone Snails or analogous to the naturally available Burroughs-Wellcome Co., and vecuronium bromide which peptides, and which preferably include two disulfide bonds. is available as Norcuron from Organon Inc.) and (ii) depo The C-conotoxins, as described herein, are useful for as larizing agents which transiently activate muscle receptors neuromuscular blocking agents, Such as muscle relaxants. and result in their blockade (e.g., decamethonium iodide, The publications and other materials used herein to illu and Succinylcholine chloride which is available as Anectine minate the background of the invention, and in particular, from Burroughs-Wellcome Co.). The effects of the depolar cases to provide additional details respecting the practice, 25 izing agents are manifested as fasciculations and flaccid are incorporated by reference, and for convenience are paralysis which are observed to occur rapidly after their referenced in the following text by author and date and are injection. listed alphabetically by author in the appended bibliography. The effects of depolarizing agents (DA) and non The predatory cone Snails (Conus) have developed a depolarizing agents (NDA) are separated based on their unique biological Strategy. Their venom contains relatively duration of action from ultrashort acting (e.g., for a depo Small peptides that are targeted to various neuromuscular larizing agent Such as Succinylcholine chloride) to interme receptors and may be equivalent in their pharmacological diate (e.g., for a non-depolarizing agent Such as atracurium diversity to the alkaloids of plants or Secondary metabolites beSylate). Certain types of muscle relaxants are useful as of microorganisms. Many of these peptides are among the neuromuscular blocking agents in clinical applications, and Smallest nucleic acid-encoded translation products having 35 have found use as adjuvants to Surgical anesthesia, in defined conformations, and as Such, they are Somewhat orthopedic Surgical procedures and in facilitating endotra unusual. Peptides in this size range normally equilibrate cheal intubation procedures. Some of these compounds (e.g., among many conformations. Proteins having a fixed con Succinylcholine chloride) are routinely used to provide muscle relaxation during Cesarean Section procedures. formation are generally much larger. 40 The cone Snails that produce these peptides are a large It is desirable for neuromuscular blocking agents to be genus of Venomous gastropods comprising approximately locally acting and highly Selective for binding to muscle 500 species. All cone Snail species are predators that inject nicotinic acetylcholine receptor Sites. AS Such, when a Venom to capture prey, and the Spectrum of animals that the patient is treated with anesthesia, the muscle relaxant is genus as a whole can envenomate is broad. A wide variety 45 applied (e.g., intravenously or by injection), in order to of hunting Strategies are used, however, every Conus Species cause the muscle to relax and hence minimize muscle uses fundamentally the same basic pattern of envenomation. contraction. Several peptides isolated from Conus venoms have been In anesthesia, neuromuscular blocking agents are used to characterized. These include the C-, u- and co-conotoxins provide skeletal muscular relaxation during Surgery and which target nicotinic acetylcholine receptors, muscle 50 during intubation of the trachea. All of the conventional Sodium channels, and neuronal calcium channels, respec nondepolarizing agents when used for producing skeletal tively (Olivera et al., 1985). Conopressins, which are vaso muscle relaxation in Surgery have a long duration of action pressin analogs, have also been identified (Cruz et al. 1987). e.g., 60 to 180 minutes in man. The depolarizing agents on In addition, peptides named conantokins have been isolated the other hand provide muscle relaxation at dosages nor from Conus geographus and Conus tulipa (Mena et al., 55 mally used for Surgery which is less than the duration of 1990; Haack et al., 1990). action of nondepolarizing agents. For example, Succinylcho The C-conotoxins are Small peptides highly specific for line provides a short duration of action of about 5 to 15 neuromuscular junction nicotinic acetylcholine receptors minutes whereas decamethonium provides about 20 to 40 (Gray et al., 1981; Marshall and Harvey, 1990; Blount et al., minutes duration of muscle relaxation. The long duration of 1992). The C-conotoxin peptides MI and GI are selective for 60 action of nondepolarizing agents is unacceptable in many the C/o Subunit interface of the neuromuscular junction Surgical procedures which take less than one hour because nicotinic receptor over the O/Y subunit interface by >10,000 the patient is not generally fully recovered from their effects fold, while the C-conotoxin peptides EI and EIA bind both e.g., the patient may be unable to breathe adequately on his Sites with equal affinity. However, none of these peptides or her own. show Significant affinity for neuronal nicotinic receptorS. 65 Each nondepolarizing agent has inherent Side-effects. For Various compounds having muscle relaxant properties are example, gallamine and pancuronium may cause set forth in U.S. Pat. Nos. 4,190,674; 4,508,715; 4,761,418; tachycardia, d-tubocurarine and diallyltoxiferine may cause US 6,268.473 B1 3 4 hypotension, and Succinylcholine may cause fasciculations, Tyr and 5-amino-Tyr), Lys, ornithine, homoargine, myalgia, potassium release, cardiovascular effects, immu N-methy-LyS, N,N-dimethyl-Lys, N.N.N-trimethyl-Lys or nological reactions and malignant hyperthermia. While Such any unnatural basic amino acid (Such as N-1-(2- drugs can be pharmacologically antagonized with anticho pyrazolinyl)-Arg), Xaas is His, ASn or halo-His; Xaa, is Pro linesterase agents, this obviously necessitates the adminis or hyroxy-Pro; Xaa, is Ala, Gly, Ser or Thr; Xaas is Gly or tration of a Second drug which itself may have its own side Ala; Xaa, is Arg, LyS, Pro, hydroxy-Pro, Gly, Gln, ornithine, effects e.g., bradycardia, gut Spasm and bronchorrhea. Thus homoargine, N-methy-Lys, N,N-dimethyl-Lys, N.N.N- to overcome the aforementioned side-effects of the anticho trimethyl-Lys or any unnatural basic amino acid (Such as linesterase agents, a third drug, an anticholinergic drug e.g., N-1-(2-pyrazolinyl)-Arg), Xaao is His, halo-His, ASn, LyS, atropine must also be given. Tyr, mono-halo-Tyr, di-halo-Tyr, O-Sulpho-Tyr, O-phospho Tyr, nitro-Tyr, N-methy-Lys, N,N-dimethyl-Lys, N.N.N- With the use of depolarizing agents, there is no need to trimethyl-LyS, Arg, homoarginine, ornithine or any unnatu reverse the effects of the depolarizing agents, in certain ral basic amino acid (such as N-1-(2-pyrazolinyl)-Arg); patients the effects are much prolonged because of abnormal Xaa is Tyr, Phe, mono-halo-Tyr, di-halo-Tyr, O-Sulpho metabolism of the agent by the patient. The polarizing Tyr, O-phospho-Tyr, nitro-Tyr, any unnatural hydroxy con agents due to the mode of action which initially causes 15 taining amino acid (Such as 4-hydroxymethyl-Phe, skeletal muscle contraction and Stimulation of Smooth 4-hydroxyphenyl-Gly, 2,6-dimethyl-Tyr and 5-amino-Tyr), muscles are also known to cause the following Side-effects Trp (D or L), halo-Trp, neo-Trp, or any unnatural aromatic in certain instances, increased intraocular, and intragastric amino acid (such as nitro-Phe, 4-substituted-Phe wherein the tension, cardiac arrhythmias, potassium release, and muscle Substituent is C-C alkyl, carboxyl, hyrdroxymethyl, pain. These side-effects caused by the depolarizing agents sulphomethyl, halo, phenyl, -CHO,-CN, -SOH and are not caused by the nondepolarizing agents. It is therefore -NHAc); Xaa is Ile, Ser, Thr, Asp, Gly, ASn, Glu, Gla or clearly evident that a new neuromuscular blocking agent Val; Xaa1 is des-Xaas, LyS, Arg, Ornithine, homoargine, having the relatively few side-effects and the reversibility of N-methy-LyS, N,N-dimethyl-Lys, N.N.N-trimethyl-Lys or the nondepolarizing agents yet being of considerably shorter any unnatural basic amino acid (Such as N-1-(2- i.e., intermediate, duration of action is needed. 25 pyrazolinyl)-Arg), Xaa1 is des-Xaa14, Gly, LyS, Arg, It is desired to provide a compound useful as a muscle ornithine, homoargine, N-methy-LyS, N,N-dimethyl-LyS, relaxant. In particular, it is desired to provide an agonist N,N,N-trimethyl-Lys or any unnatural basic amino acid which has activity at relatively low concentrations as a (Such as N-1-(2-pyrazolinyl)-Arg), Xaas is des-Xaas, Gly, neuromuscular blocking agent. It is also desired to achieve Thr, Ser, His, halo-His, LyS, Arg, Ornithine, homoargine, muscle relaxation at concentrations of agonist that are N-methy-LyS, N,N-dimethyl-Lys, N.N.N-trimethyl-Lys or devoid of any ganglionic effects (e.g., So as to not exhibit any unnatural basic amino acid (Such as N-1-(2- Side effects Such as those associated with interaction with pyrazolinyl)-Arg), Xaa, is des-Xaa, Ser or Thr, Xaa, is cardiovascular sites). AS Such, it is desired to provide muscle des-Xaa, or Cys, Xaas is des-Xaas, Ser or Thr, Xaa, is relaxant compositions and methods for providing muscle des-Xaao, Arg, LyS, ornithine, homoargine, N-methy-LyS, relaxation. Finally, it is desired to identify additional 35 N,N-dimethyl-Lys, N,N,N-trimethyl-Lys or any unnatural C-conotoxin peptides for use as neuromuscular blocking basic amino acid (Such as N-1-(2-pyrazolinyl)-Arg), Xaao agents. is des-Xaa, Thr, Ser, Pro or hydroxy-Pro; Xaa, is des Xaa, Leu, Ser or Thr, Xaa- is des-Xaa, Glu or Gla; SUMMARY OF THE INVENTION Xaa- is des-Xaa, Pro or hydroxy-Pro; Xaa is des-Xaa, The invention relates to relatively short peptides (termed 40 Arg, LyS, ornithine, homoargine, N-methy-LyS, N,N- C-conotoxins herein), about 10–25 residues in length, which dimethyl-Lys, N.N.N-trimethyl-Lys or any unnatural basic are naturally available in minute amounts in the Venom of amino acid (Such as N-1-(2-pyrazolinyl)-Arg); and Xaas is the cone Snails or analogous to the naturally available des-Xaas, Arg, LyS, ornithine, homoargine, N-methy-LyS, peptides, and which preferably include two disulfide bonds. 45 N,N-dimethyl-Lys, N,N,N-trimethyl-Lys or any unnatural The C-conotoxins, as described herein, are useful for as basic amino acid (Such as N-1-(2-pyrazolinyl)-Arg). The neuromuscular blocking agents, Such as muscle relaxants, C-terminus may contain a free carboxyl group or an amide for treating benign eSSential blepharospasm and other forms group, preferably an amide group. The halo is chlorine, of focal dystonia and for anti-wrinkle use. bromine or iodine, preferably iodine for Tyr and bromine for More Specifically, the present invention is directed to the 50 Trp. The Cys residues may be in D or L configuration and neuromuscular blocking use of C-conotoxin peptides of two may optionally be Substituted with homocysteine. classes, namely, (a) C3/5 or C.3/6 and (b) C4/7, as described Useful peptides include GI (Gray et al., 1981), GIA (Gray herein. The first class of C-conotoxin peptides has the et al., 1981), GII (Gray et al., 1981), MI (McIntosh et al., general formula I: 1982), SI (Zafaralla et al., 1988), SIA (Myers et al., 1991), Xaa-Xaa-Xaa-Xaa-CyS-CyS-Xaas-Xaa-Xaa-Cys 55 SIB (same as SI, except further contains Glu at N-terminus), Xaas-Xaao-Xaa1-Xaa1-Xaa1-Xaa-CyS-Xaa1-Xaas SII (Olivera et al., 1996), SIIA (Olivera et al., 1996), RI Xaa16-Xaa17-Xaa1s-Xaa1o-Xaa2o-Xaa 21-Xaa-22-Xaa23 (same as G1, except Tyr for Lys), R1.3 (below), R1.4 Xaa-Xaas (SEQ ID NO:1), wherein Xaa is des-Xaa1 or (below), Sm1.1 (below), S11 (below), S2 (below); GIB Gly, Xaa- is des-Xaa, ASn, Arg, Asp, Ser, Thr, LyS, (same as R1); MnII (below); A1.2 (below); A1.3 (below); ornithine, homoargine, N-methy-LyS, N,N-dimethyl-LyS, 60 A1.7 (below); A1.8 (below); Ay1.1 (below); Ay1.1a (below); N,N,N-trimethyl-Lys or any unnatural basic amino acid M1.1 (below); M1.3 (below); M1.4 (below); M1.5 (below); (Such as N-1-(2-pyrazolinyl)-Arg), Xaa- is des-Xaa, (Gly, O1.3 (below); S1.3 (below); Sa (below). Glu or Y-carboxy-Glu (Gla); Xaa, is des-Xaa, Glu, Gla, The Second class of C-conotoxin peptides has the general Gln, pyro-Glu, Arg, Ile Tyr, mono-halo-Tyr, di-halo-Tyr, formula II: O-Sulpho-Tyr, O-phospho-Tyr, nitro-Tyr, Cys, His, halo-His, 65 Xaa-Xaa-Xaa-CyS-Cys-Xaa-Xaas-Xaa-Xaa-CyS any unnatural hydroxy containing amino acid (Such as Xaas-Xaao-Xaao-Xaa1-Xaa-Xaa1-Ile-Cys-Xaa1-Xaa1 4-hydroxymethyl-Phe, 4-hydroxyphenyl-Gly, 2,6-dimethyl Xaas (SEQ ID NO:2), wherein, Xaa is des-Xaa, Arg, Ser,

US 6,268.473 B1 7 8 M1.4: SEQ ID NO:17, wherein Xaa is Pro, Xaa, is Tyr amino acids and aromatic amino acids and pages 66-87 for and Xaa, is LyS, basic amino acids; See also http://www.amino-acids.com), incorporated herein by reference, by and available from RSP M1.5: SEQ ID NO:18, wherein Xaa, is Pro, Xaa, is Tyr Amino Acid Analogues, Inc., Worcester, Mass. and Xaa is LyS, Optionally, in the peptides of general formulas I and II O1.3: SEQ ID NO:19, wherein Xaa is Pro, Xaa, is Tyr, and the Specific peptides described above, the ASn residues Xaa is Lys and Xaas is Gln; may be modified to contain an N-glycan and the Ser and Thr S1.3: SEQ ID NO:20, wherein Xaa, is Pro, Xaa, is Tyr, residues may be modified to contain an O-glycan. In accor Xaa is Lys and Xaas is Gln; and dance with the present invention, a glycan shall mean any Sa: SEQ ID NO:21, wherein Xaa- is Pro, Xaa is Tyr and N-, S- or O-linked mono-, di-, tri-, poly- or oligosaccharide Xaa is Lys. that can be attached to any hydroxy, amino or thiol group of The C-terminus is preferably amidated in each of these natural or modified amino acids by Synthetic or enzymatic Specific peptides. methodologies known in the art. The monosaccharides mak The present invention is also directed to novel Specific ing up the glycan can include D-allose, D-altrose, C-conotoxin peptides of class II having the formulas: 15 D-glucose, D-mannose, D-gulose, D-idose, D-galactose, Arg-Asp-Xaa-CyS-CyS-Ser-Asn-Xaa-Val-Cys-Thr-Val D-talose, D-galactosamine, D-glucosamine, D-N-acetyl His-Asn-Xaa-Gln-Ile-Cys (SEQ ID NO:22); glucosamine (GlcNAc), D-N-acetyl-galactosamine Arg-Ala-CyS-CyS-Ser-Xaa-Xaa-Xaa-CyS-ASn-Val (GalNAc), D-fucose or D-arabinose. These saccharides may be structurally modified, e.g., with one or more O-Sulfate, Asn-Xaa-Xaa-Xaa-Ile-Cys (SEQ ID NO:23); O-phosphate, O-acetyl or acidic groups, Such as Sialic acid, Gly-Gly-Cys-CyS-Ser-Xaa-Xaa-Xaa-Cys-Asn-Val including combinations thereof. The gylcan may also Ser-Xaa-Xaa-Xaa-Ile-Cys (SEQ ID NO:24); include Similar polyhydroxy groups, Such as CyS-CyS-Ser-Xaa-Xaa-Xaa-CyS-ASn-Val-Ser-Xaa D-penicillamine 2.5 and halogenated derivatives thereof or Xaa-Xaa-Ile-Cys (SEQ ID NO:25); polypropylene glycol derivatives. The glycosidic linkage is Ala-CyS-CyS-Ser-Xaa-Xaa-Xaa-Cys-Asn-Val-Asn 25 beta and 1-4 or 1-3, preferably 1-3. The linkage between Xaa-Xaa-Xaa-Ile-Cys-Gly-Gly-Arg (SEQ ID the glycan and the amino acid may be alpha or beta, NO:26); and preferably alpha and is 1-. Ser-Leu-Leu-Cys-Cys-Thr-Ile-Xaa-Ser-Cys-Xaa-Ala Core O-glycans have been described by Van de Steen et Ser-Xaa-Xaa-Asp-Ile-Cys (SEQ ID NO:27), al. (1998), incorporated herein by reference. Mucin type wherein Xaa is Glu or Y-carboxy-Glu (Gla); Xaa is Pro or O-linked oligosaccharides are attached to Ser or Thr (or hydroxy-Pro; Xaa is Tyr, mono-halo-Tyr, di-halo-Tyr, other hydroxylated residues of the present peptides) by a O-Sulpho-Tyr, O-phospho-Tyr or nitro-Tyr; Xaa, is Lys, GalNAc residue. The monosaccharide building blocks and N-methyl-LyS, N,N-dimethyl-Lys or N.N,N-trimethyl-Lys; the linkage attached to this first GalNAc residue define the and the C-terminus contains a carboxyl or amide group, “core glycans,” of which eight have been identified. The preferably an amide group. The halo is preferably chlorine 35 type of glycosidic linkage (orientation and coinectivities) are or iodine, more preferably iodine. In addition, the His defined for each core glycan. Suitable glycans and glycan residues may be substituted with halo-His; the Arg residues analogs are described further in U.S. Ser. No. 09/420,797, may be Substituted by LyS, ornithine, homoargine, N-methy filed Oct. 19, 1999 and in PCT Application No. PCT/US99/ Lys, N,N-dimethyl-Lys, N,N,N-trimethyl-Lys or any 24380, filed Oct. 19, 1999, both incorporated herein by unnatural basic amino acid (Such as N-1-(2-pyrazolinyl)- 40 reference. A preferred glycan is Gal(B1->3)GalNAc(C.1->). Arg); the Lys residues may be Substituted by Arg, ornithine, Optionally, in the peptides of general formulas I and II homoargine, N-methy-Lys, N,N-dimethyl-Lys, N.N.N- and the Specific peptides described above, pairs of CyS trimethyl-Lys or any unnatural basic amino acid (Such as residues may be replaced pairwise with LyS-Glu pairs. N-1-(2-pyrazolinyl)-Arg); and the Tyr residues may be Sub Sequential coupling by known methods (Bamay et al., 2000; Stituted with any unnatural hydroxy containing amino acid 45 Hruby et al., 1994; Bitan et al., 1997) allows replacement of (such as 4-hydroxymethyl-Phe, 4-hydroxyphenyl-Gly, 2,6- native cyS bridges with lactam bridges. dimethyl-Tyr and 5-amino-Tyr). The present invention is further directed to propeptides More Specifically, the present invention is directed to the and nucleic acid Sequences encoding the propeptides or following C-conotoxin peptides of class II: peptides as described in further detail herein. P1.2: SEQ ID NO:22, wherein Xaa, is Pro; 50 BRIEF DESCRIPTION OF THE FIGURES P1.3: SEQ ID NO:23, wherein Xaa is Glu, Xaa is Pro FIG. 1 shows onset and recovery time of neuromuscular and Xaa is Tyr; block for different doses (87, 100 or 150 tug/kg) of the Sl1.4: SEQ ID NO:24, wherein Xaa is Glu, Xaa is Pro C-conotoxin peptide MI. and Xaa is Tyr; 55 FIG. 2 shows onset and recovery time of neuromuscular Sl1.4A: SEQ ID NO:25, wherein Xaa is Glu, Xaa is Pro block for different doses (180, 217 or 250 ug/kg) of and Xaa is Tyr; C-conotoxin peptide GI. S11.8: SEQ ID NO:26, wherein Xaa is Glu, Xaa, is Pro FIG. 3 shows dose response curves for the C-conotoxin and Xaa is Tyr; and peptides MI (O) and GI (). Ta: SEQ ID NO:27, wherein Xaa- is Pro, Xaa is Tyr and 60 FIG. 4 shows onset and recovery time of neuromuscular Xaa is Lys. block for different doses (18.76, 28.125, 37.5, 75 or 150 The C-terminus is preferably amidated in each of these Aug/kg) of the C-conotoxin peptide mono-iodo-Tyr-MI. Specific peptides. FIG. 5 shows onset and recovery time of neuromuscular The above and other unnatural basic amino acids, unnatu block for different doses (125, 137.5 or 150 lug/kg) of the ral hydroxy containing amino acids or unnatural aromatic 65 C-conotoxin peptide di-iodo-Tyr-MI. amino acids are described in Building Block Index, Version FIG. 6 shows dose response curve for the C-conotoxin 3.0 (1999 Catalog, pages 4-47 for hydroxy containing peptide mono-iodo-Tyr-MI. US 6,268.473 B1 10 FIG. 7 shows dose response curve for the C-conotoxin NO:58 is a DNA sequence coding for the MnI propeptide. peptide di-iodo-Tyr-MI. SEQ ID NO:59 is the amino acid sequence of the MnI propeptide. SEQ ID NO:60 is a DNA sequence coding for SUMMARY OF THE SEQUENCE LISTING the Cr1.1 propeptide. SEQ ID NO:61 is the amino acid SEQ ID NO:1 is a generic formula for C-conotoxin sequence of the Cr1.1 propeptide. SEQ ID NO:62 is a DNA peptides of Class I useful as neuromuscular blocking agents. sequence coding for the R1.2 propeptide. SEQ ID NO:63 is SEQ ID NO:2 is a generic formula for C-conotoxin peptides the amino acid sequence of the R1.2 propeptide. SEQ ID of Class II useful as neuromuscular blocking agents. SEQID NO:64 is a DNA sequence coding for the A1.3 propeptide. NO:3 is a generic formula for the peptide R1.3. SEQ ID SEQ ID NO:65 is the amino acid sequence of the A1.3 NO:4 is a generic formula for the peptide R1.4. SEQ ID propeptide. SEQ ID NO:66 is a DNA sequence coding for NO:5 is a generic formula for the peptide Sm1.1. SEQ ID the A1.7 propeptide. SEQ ID NO:67 is the amino acid NO:6 is a generic formula for the peptide S11. SEQID NO:7 sequence of the A1.7 propeptide. SEQ ID NO:68 is a DNA is a generic formula for the peptide S2. SEQ ID NO:8 is a sequence coding for the A1.8 propeptide. SEQ ID NO:69 is generic formula for the peptide MnII. SEQ ID NO:9 is a the amino acid sequence of the A1.8 propeptide. SEQ ID generic formula for the peptide A1.2. SEQ ID NO:10is a 15 NO:70 is a DNA sequence coding for the Ay1.1 propeptide. generic formula for the peptide A1.3. SEQ ID NO:11 is SEQ ID NO:71 is the amino acid sequence of the Ay1.1 ageneric formula forthe peptide A1.7. SEQ ID NO:12 is a propeptide. SEQ ID NO:72 is a DNA sequence coding for generic formula for the peptide A1.8. SEQ ID NO:13 is a the Ay1.1a propeptide. SEQ ID NO:73 is the amino acid generic formula for the peptide Ay 1.1. SEQ ID NO:14 is a sequence of the Ay1.1a propeptide. SEQID NO:74 is a DNA generic formula for the peptide Ay1.1a. SEQ ID NO:15 is a sequence coding for the M1.1 propeptide. SEQID NO:75 is generic formula for the peptide M1.1. SEQ ID NO:16 is a the amino acid sequence of the M1.1 propeptide. SEQ ID generic formula for the peptide M1.3. SEQ ID NO:17 is a NO:76 is a DNA sequence coding for the M1.3 propeptide. generic formula for the peptide M1.4. SEQ ID NO:18 is a SEQ ID NO:77 is the amino acid sequence of the M1.3 generic formula for the peptide M1.5. SEQ ID NO:19 is a propeptide. SEQ ID NO:78 is a DNA sequence coding for generic formula for the peptide O1.3. SEQ ID NO:20 is a 25 the M1.4 propeptide. SEQ ID NO:79 is the amino acid generic formula for the peptide S1.3. SEQ ID NO:21 is a sequence of the M1.4 propeptide. SEQ ID NO:80 is a DNA generic formula for the peptide Sa. SEQ ID NO:22 is a sequence coding for the M1.5 propeptide. SEQID NO:81 is generic sequence for the peptide P1.2. SEQ ID NO:23 is a the amino acid sequence of the M1.5 propeptide. SEQ ID generic sequence for the peptide P1.3. SEQ ID NO:24 is a NO:82 is a DNA sequence coding for the O1.3 propeptide. generic sequence for the peptide S11.4. SEQ ID NO:25 is a SEQ ID NO:83 is the amino acid sequence of the O1.3 generic sequence for the peptide S11.4A. SEQ ID NO:26 is propeptide. SEQ ID NO:84 is a DNA sequence coding for a generic sequence for the peptide S11.8. SEQ ID NO:27 is the S1.3 propeptide. SEQ ID NO:85 is the amino acid a generic formula for the peptide Ta. SEQ ID NO:28 is a sequence of the S1.3 propeptide. SEQ ID NO:85 is a DNA DNA sequence coding for the GI propeptide. SEQID NO:29 sequence coding for the EI propeptide. SEQID NO:87 is the is the amino acid sequence of the GI propeptide. SEQ ID 35 amino acid sequence of the EI propeptide. SEQ ID NO:88 NO:30 is a DNA sequence coding for the SIB propeptide. is a DNA sequence coding for the EIA propeptide. SEQ ID SEQ ID NO:31 is the amino acid sequence of the SIB NO:89 is the amino acid sequence of the EIA propeptide. propeptide. SEQ ID NO:32 is a DNA sequence coding for SEQ ID NO:90 is a DNA sequence coding for the P1.2 the R1 propeptide. SEQ ID NO:33 is the amino acid propeptide. SEQID NO:91 is the amino acid sequence of the sequence of the R1 propeptide. SEQ ID NO:34 is a DNA 40 P1.2 propeptide. SEQ ID NO:92 is a DNA sequence coding sequence coding for the R1.3 propeptide. SEQ ID NO:35 is for the P1.3 propeptide. SEQ ID NO:93 is the amino acid the amino acid sequence of the R1.3 propeptide. SEQ ID sequence of the P1.3 propeptide. SEQ ID NO:94 is a DNA NO:36 is a DNA sequence coding for the R1.4 propeptide. sequence coding for the S11.4 propeptide. SEQ ID NO:95 is SEQ ID NO:37 is the amino acid sequence of the R1.4 the amino acid sequence of the S11.4 propeptide. SEQ ID propeptide. SEQ ID NO:38 is a DNA sequence coding for 45 NO:96 is a DNA sequence coding for the S11.4A propeptide. the Sm1.1 propeptide. SEQ ID NO:39 is the amino acid SEQ ID NO:97 is the amino acid sequence of the S11.4A sequence of the Sm1.1 propeptide. SEQID NO:40 is a DNA propeptide. SEQ ID NO: 98 is a DNA sequence coding for sequence coding for the SIIA propeptide. SEQ ID NO:41 is the S11.8 propeptide. SEQ ID NO:99 is the amino acid the amino acid sequence of the SIIA propeptide. SEQ ID sequence of the S11.8 propeptide. SEQID NO:100 is a DNA NO:42 is a DNA sequence coding for the S11 peptide. SEQ 50 sequence coding for the P1.1 propeptide. SEQID NO:101 is ID NO:43 is the amino acid sequence of the S11 peptide. the amino acid Sequence of the P1.1 propeptide. SEQ ID NO:44 is a DNA sequence coding for the S2 peptide. SEQID NO:45 is the amino acid sequence of the S2 DETAILED DESCRIPTION OF THE peptide. SEQ ID NO:46 is a DNA sequence coding for the PREFERRED EMBODIMENT GIB propeptide. SEQ ID NO:47 is the amino acid sequence 55 The invention relates to relatively short peptides (termed of the GIB propeptide. SEQ ID NO:48 is a DNA sequence C-conotoxins herein), about 10–25 residues in length, which coding for the Mini propeptide. SEQID NO:49 is the amino are naturally available in minute amounts in the Venom of acid sequence of the Mn(I propeptide. SEQ ID NO:50 is a the cone Snails or analogous to the naturally available DNA sequence coding for the A1.2 propeptide. SEQID NO: peptides, and which preferably include two disulfide bonds. 51 is the amino acid Sequence of the A1.2 propeptide. SEQ 60 The C-conotoxins, as described herein, are useful for as ID NO:52 is a DNA sequence coding for the A1.1 propep neuromuscular blocking agents, Such as muscle relaxants, tide. SEQ ID NO:53 is the amino acid sequence of the A1.1 for treating benign eSSential blepharospasm and other forms propeptide. SEQ ID NO:54 is a DNA sequence coding for of focal dystonia and for anti-wrinkle use. the Bt1.6 propeptide. SEQ ID NO:55 is the amino acid In one aspect, the present invention relates to a method for sequence of the Bt1.6 propeptide. SEQ ID NO:56 is a DNA 65 providing relaxation of muscle. The method involves admin sequence coding for the Cn 1.1 propeptide. SEQ ID NO:57 istering to a patient an effective amount of an O-conotoxin is the amino acid sequence of the Cn 1.1 propeptide. SEQ ID peptide having the general formula Set forth above. Exem US 6,268.473 B1 11 12 plary methods involve administering to a patient an effective esters of N-hydroxyphthalimide or N-hydroxy-succinimide, amount of MI, GI, El, mono-iodo-MI (Tyra of MI having an and the various cleavage reagents, to carry out reaction in iodine) or di-iodo-MI (Tyra of MI having two iodines). Solution, with Subsequent isolation and purification of The present invention, in another aspect, relates to a intermediates, is well known classical peptide methodology. pharmaceutical composition comprising an effective amount Classical Solution Synthesis is described in detail in the of an O-conotoxin peptide having the general formula Set treatise, “Methoden der Organischen Chemie (Houben forth above. Such a pharmaceutical composition has the Weyl): Synthese von Peptiden,” (1974). Techniques of capability of acting as a neuromuscular non-depolarizing exclusively Solid-phase Synthesis are set forth in the agent, and hence has the capability of acting as a muscle textbook, “Solid-Phase Peptide Synthesis,” (Stewart and relaxant. Exemplary pharmaceutical compositions acting as Young, 1969), and are exemplified by the disclosure of U.S. Pat. No. 4,105,603 (Vale et al., 1978). The fragment con neuromuscular non-depolarizing muscle relaxants include as densation method of synthesis is exemplified in U.S. Pat. an active ingredient MI, GI, El, mono-iodo-MI or di-iodo No. 3,972,859 (1976). Other available syntheses are exem MI. plified by U.S. Pat. Nos. 3,842,067 (1974) and 3,862,925 The C-conotoxin peptides described herein are Suffi (1975). The synthesis of peptides containing ciently Small to be chemically Synthesized. General chemi 15 Y-carboxyglutamic acid residues is exemplified by Rivier et cal Syntheses for preparing the foregoing C-conotoxin pep al. (1987), Nishiuchi et al. (1993) and Zhou et al. (1996). tides are described hereinafter. Various ones of the Common to Such chemical Syntheses is the protection of C-conotoxin peptides can also be obtained by isolation and the labile Side chain groups of the various amino acid purification from Specific Conus Species using the technique moieties with Suitable protecting groups which will prevent described in U.S. Pat. No. 4,447,356 (Olivera et al., 1984), a chemical reaction from occurring at that Site until the the disclosure of which is incorporated herein by reference. group is ultimately removed. Usually also common is the Although the C-conotoxin peptides of the present inven protection of an O-amino group on an amino acid or a tion can be obtained by purification from cone Snails, fragment while that entity reacts at the carboxyl group, because the amounts of C-conotoxin peptides obtainable 25 followed by the Selective removal of the C-amino protecting from individual Snails are very Small, the desired Substan group to allow Subsequent reaction to take place at that tially pure C-conotoxin peptides are best practically location. Accordingly, it is common that, as a step in Such a obtained in commercially valuable amounts by chemical Synthesis, an intermediate compound is produced which Synthesis using Solid-phase Strategy. For example, the yield includes each of the amino acid residues located in its from a single cone Snail may be about 10 micrograms or leSS desired Sequence in the peptide chain with appropriate of C-conotoxin peptide. By “Substantially pure' is meant Side-chain protecting groups linked to various ones of the that the peptide is present in the Substantial absence of other biological molecules of the same type; it is preferably residues having labile Side chains. present in an amount of at least about 85% purity and AS far as the Selection of a side chain amino protecting preferably at least about 95% purity. Chemical synthesis of group is concerned, generally one is chosen which is not 35 removed during deprotection of the C-amino groups during biologically active C-conotoxin peptides depends of course the Synthesis. However, for Some amino acids, e.g., His, upon correct determination of the amino acid Sequence. protection is not generally necessary. In Selecting a particu The C-conotoxin peptides can also be produced by recom lar Side chain protecting group to be used in the Synthesis of binant DNA tecliniques well known in the art. Such tech the peptides, the following general rules are followed: (a) the niques are described by Sambrook et al. (1989). The pep 40 protecting group preferably retains its protecting properties tides produced in this manner are isolated, reduced if and is not split off under coupling conditions, (b) the necessary, and oxidized to form the correct disulfide bonds. protecting group should be stable under the reaction condi One method of forming disulfide bonds in the conantokin tions Selected for removing the C-amino protecting group at peptides of the present invention is the air oxidation of the each step of the Synthesis, and (c) the side chain protecting linear peptides for prolonged periods under cold room 45 group must be removable, upon the completion of the temperatures or at room temperature. This procedure results Synthesis containing the desired amino acid Sequence, under in the creation of a Substantial amount of the bioactive, reaction conditions that will not undesirably alter the peptide disulfide-linked peptides. The oxidized peptides are frac chain. tionated using reverse-phase high performance liquid chro It should be possible to prepare many, or even all, of these matography (HPLC) or the like, to separate peptides having 50 peptides using recombinant DNA technology. However, different linked configurations. Thereafter, either by com when peptides are not So prepared, they are preferably paring these fractions with the elution of the native material prepared using the Merrifield Solid-phase Synthesis, or by using a simple assay, the particular fraction having the although other equivalent chemical Syntheses known in the correct linkage for maximum biological potency is easily art can also be used as previously mentioned. Solid-phase determined. However, because of the dilution resulting from 55 Synthesis is commenced from the C-terminus of the peptide the presence of other fractions of less biopotency, a Some by coupling a protected C.-amino acid to a Suitable resin. what higher dosage may be required. Such a starting material can be prepared by attaching an The peptides are Synthesized by a Suitable method, Such C.-amino-protected amino acid by an ester linkage to a as by exclusively Solid-phase techniques, by partial Solid chloromethylated resin or a hydroxymethyl resin, or by an phase techniques, by fragment condensation or by classical 60 amide bond to a benzhydrylamine (BHA) resin or param Solution couplings. ethylbenzhydrylamine (MBHA) resin. Preparation of the In conventional Solution phase peptide Synthesis, the hydroxymethyl resin is described by Bodansky et al. (1966). peptide chain can be prepared by a Series of coupling Chloromethylated resins are commercially available from reactions in which constituent amino acids are added to the Bio Rad Laboratories (Richmond, Calif.) and from Lab. growing peptide chain in the desired Sequence. Use of 65 Systems, Inc. The preparation of Such a resin is described by various coupling reagents, e.g., dicyclohexylcarbodiimide or Stewart and Young (1969). BHA and MBIHA resin supports diisopropylcarbonyldimidazole, various active esters, e.g., are commercially available, and are generally used when the US 6,268.473 B1 13 14 desired polypeptide being Synthesized has an unsubstituted all remaining Side chain protecting groups and also the amide at the C-terminus. Thus, Solid resin Supports may be C.-amino protecting group at the N-terminus if it was not any of those known in the art, Such as one having the previously removed to obtain the peptide in the form of the formulae -O-CH-resin support, -NH BHA resin free acid. If Met is present in the Sequence, the Boc support, or -NH-MBHA resin support. When the unsub protecting group is preferably first removed using trifluoro stituted amide is desired, use of a BHA or MBHA resin is acetic acid (TFA)/ethanedithiol prior to cleaving the peptide preferred, because cleavage directly gives the amide. In case from the resin with HF to eliminate potential S-alkylation. the N-methyl amide is desired, it can be generated from an When using hydrogen fluoride or TFA for cleaving, one or N-methyl BHA resin. Should other substituted amides be more ScavengerS Such as aniSole, creSol, dimethyl Sulfide desired, the teaching of U.S. Pat. No. 4,569,967 (Kornreich and methylethyl Sulfide are included in the reaction vessel. et al., 1986) can be used, or should still other groups than the Cyclization of the linear peptide is preferably affected, as free acid be desired at the C-terminus, it may be preferable opposed to cyclizing the peptide while a part of the peptido to Synthesize the peptide using classical methods as Set forth resin, to create bonds between CyS residues. To effect Such in the Houben-Weyl text (1974). a disulfide cyclizing linkage, fully protected peptide can be The C-terminal amino acid, protected by Boc or Fmoc and 15 cleaved from a hydroxymethylated resin or a chloromethy by a Side-chain protecting group, if appropriate, can be first lated resin Support by ammonolysis, as is well known in the coupled to a chloromethylated resin according to the pro art, to yield the fully protected amide intermediate, which is cedure set forth in K. Horiki et al. (1978), using KF in DMF thereafter Suitably cyclized and deprotected. Alternatively, at about 60° C. for 24 hours with stirring, when a peptide deprotection, as well as cleavage of the peptide from the having free acid at the C-terminus is to be Synthesized. above resins or a benzhydrylamine (BHA) resin or a meth Following the coupling of the BOC-protected amino acid to ylbenzhydrylamine (MBHA), can take place at 0° C. with the resin Support, the C-amino protecting group is removed, hydrofluoric acid (HF) or TFA, followed by oxidation as as by using trifluoroacetic acid (TFA) in methylene chloride described above. or TFA alone. The deprotection is carried out at a tempera The peptides are also synthesized using an automatic ture between about 0° C. and room temperature. Other 25 Synthesizer. Amino acids are Sequentially coupled to an Standard cleaving reagents, Such as HCl in dioxane, and MBHA Rink resin (typically 100 mg of resin) beginning at conditions for removal of Specific C.-amino protecting the C-terminus using an Advanced Chemtech 357 Automatic groups may be used as described in Schroder & Lubke Peptide Synthesizer. Couplings are carried out using 1,3 (1965). -diisopropylcarbodimide in N-methylpyrrolidinone (NMP) After removal of the C-amino-protecting group, the or by 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium remaining C.-amino- and Side chain-protected amino acids hexafluorophosphate (HBTU) and diethylisopro are coupled Step-wise in the desired order to obtain the pylethylamine (DIEA). The FMOC protecting group is intermediate compound defined hereinbefore, or as an alter removed by treatment with a 20% solution of piperidine in native to adding each amino acid separately in the Synthesis, dimethylformamide(DMF). Resins are subsequently washed Some of them may be coupled to one another prior to 35 with DMF (twice), followed by methanol and NMP. addition to the Solid phase reactor. Selection of an appro The compounds described herein are used as neurmuscu priate coupling reagent is within the skill of the art. Particu lar blocking agents in conjunction with Surgery or for larly Suitable as a coupling reagent is N,N'- intubation of the trachea by conventional parenteral admin dicyclohexylcarbodiimide (DCC, DIC, HBTU, HATU, istration e.g., intramuscular or intravenous administration in TBTU in the presence of HoBt or HoAt). 40 Solution. Thus, the present invention relates to a method for The activating reagents used in the Solid phase Synthesis treating a patient during Surgical procedures requiring anes of the peptides are well known in the peptide art. Examples thesia and musculoskeletal relaxation. In particular, the of Suitable activating reagents are carbodiimides, Such as method comprises administering to the patient an amount of N,N'-diisopropylcarbodiimide and N-ethyl-N'-(3- a compound effective for providing relaxation of muscle. dimethylaminopropyl)carbodiimide. Other activating 45 The method involves administering an effective amount of a reagents and their use in peptide coupling are described by compound Selected from the general formulae which are Set Schroder & Lubke (1965) and Kapoor (1970). forth hereinbefore. The present invention relates to a phar Each protected amino acid or amino acid Sequence is maceutical composition incorporating a compound introduced into the Solid-phase reactor in about a twofold or 50 described herein or its pharmaceutically acceptable Salts. more excess, and the coupling may be carried out in a The manner in which the compounds are administered can medium of dimethylformamide (DMF):CHCl (1:1) or in vary. Although it is possible to administer the compound in DMF or CHCl alone. In cases where intermediate cou the form of a bulk active chemical, it is preferred to present pling occurs, the coupling procedure is repeated before the compound in the form of a pharmaceutical composition removal of the C-amino protecting group prior to the cou 55 or formulation for parenteral administration. Pharmaceutical pling of the next amino acid. The Success of the coupling compositions containing a compound of the present inven reaction at each Stage of the Synthesis, if performed tion as the active ingredient can be prepared according to manually, is preferably monitored by the ninhydrin reaction, conventional pharmaceutical compounding techniques. See, as described by Kaiser et al. (1970). Coupling reactions can for example, Remington's Pharmaceutical Sciences, 18th be performed automatically, as on a Beckman 990 automatic 60 Ed. (1990, Mack Publishing Co., Easton, Pa.). Typically, an Synthesizer, using a program Such as that reported in Rivier amount of active ingredient effective to provide muscle et al. (1978). relaxation will be admixed with a pharmaceutically accept After the desired amino acid Sequence has been able carrier. completed, the intermediate peptide can be removed from The pharmaceutical composition also can include various the resin Support by treatment with a reagent, Such as liquid 65 other components as additives or adjuncts. Exemplary phar hydrogen fluoride or TFA (if using Fmoc chemistry), which maceutically acceptable components or adjuncts include not only cleaves the peptide from the resin but also cleaves anesthetics, preservatives, antioxidants, bacterioStatic US 6,268.473 B1 15 16 agents, buffering agents, analgesics, anti-inflammatory compounds injected intravenously generally is from about agents, anti-pyretics, Stabilizing agents, thickening agents 0.001 mg/kg to about 0.5 mg/kg body weight, preferably and Suspending agents. Such components can provide addi from about 0.01 mg/kg to about 0.4 mg/kg, and more tional therapeutic benefit, or act towards preventing any preferably from about 0.01 mg/kg to about 0.25 mg/kg. potential side effects which may be posed as a result of Following administration of typical compounds in Such a administration of the pharmaceutical composition. concentration range, the onset of paralysis normally devel Typically, the pharmaceutical composition is adminis opS within 1 to 2 minutes, and persists for a short period of tered as an aqueous or non-aqueous Solution, as a time before recovery is achieved. For infants and children Suspension, or as an emulsion in a pharmaceutically accept undergoing long Surgical procedures, the effective dose of able liquid or mixture of liquids. The compound within the typical compounds is administered through continuous or pharmaceutical composition is administered internally by intermittent intravenous perfusion at a rate from about 0.001 injection or intravenously. For example, the pharmaceutical mg/min to aobut 0.5 mg/min, preferably from about 0.005 composition can be administered intravenously as an infu mg/min to about 0.3 mg/min, and more preferably from Sion (e.g., within aqueous dextrose or saline Solutions). about 0.005 mg/min to about 0.2 mg/min. The total amount Exemplary methods for administering Such muscle relax 15 of drug administered using Such a parenteral route of admin ant compounds (e.g., So as to achieve Sterile or aseptic istration generally does not exceed a total of 10 mg, often conditions) will be apparent to the skilled artisan. Certain does not exceed 5 mg and frequently does not exceed 2 mg. methods Suitable for administering compounds useful Following administration of typical compounds in the Speci according to the present invention are Set forth in Goodman fied amounts, the onset of paralysis typically develops and Gilman's The Pharmacological Basis of Therapeutics, within 1 to 2 minutes and persists for the duration of the 7th Ed. (1985). The administration to the patient can be Superfusion. intermittent; or at a gradual, continuous, constant or con Such formulations are normally presented in unit dosage trolled rate. Administration can be to a warm-blooded ani forms Such as ampoules or disposable injection devices, or mal (e.g. a mammal, Such as a mouse, rat, cat, rabbit, dog, in multidose forms Such as a bottle from which the appro pig, cow or monkey); but advantageously is administered to 25 priate dose may be withdrawn. All such formulations should a human being. Administration occurs after general anes be rendered sterile. thesia is administered. The frequency of administration The compounds of this invention may be presented as a normally is determined by an anesthesiologist, and typically powder e.g., as a unit dose in a Sealed Vial to which Sterile varies from patient to patient. water may be added by a needle, e.g., through a Seal thereof The dose of the compound is that amount effective to (Such as rubber). A Suitable unit dose to obtain a neuromus provide a desired effect for a desired time frame. By cular block for mammals is about 1 mg to 100 mg and most “effective amount' or “effective dose” is meant that amount preferably 3 to 50 mg. Thus a suitable pharmaceutical parenterally administered (e.g., injected intravenously) Suf parenteral preparation will preferably contain 20 to 100 mg ficient to bind to relevant receptor Sites on the musculosk of the compounds described herein in Solution. A pharma eletal fiber of the patient, and to elicit neuropharmacological 35 ceutical formulation may conventional contain 5 to 400 mg, effects (e.g., elicit brief depolarization, thus resulting in or 10 to 400 mg, and most preferably 5 to 200 mg of the effective short duration relaxation of skeletal muscle). Short compounds of this invention. A simple and preferred for duration typically ranges from about 5 to about 60 minutes. mulation is a Solution of a compound described herein in water which may be prepared by Simply dissolving the An effective amount of the compound administered to a 40 patient provides rapid onset and short-lived muscle relax compound into previously Sterilized pure, i.e., pyrogen free ation. For adult human patients undergoing short Surgical water under aseptic conditions and Sterilizing the Solution. procedures, the effective dose of typical compounds injected The compounds described herein may also be administered as an infusion of a dextrose Solution or a Saline Solution e.g., intravenously generally is from about 0.001 mg/kg to about Ringers Solution. 0.8 mg/kg body weight, preferably from about 0.05 mg/kg 45 to about 0.5 mg/kg, and more preferably from about 0.05 mg/kg to about 0.3 mg/kg. Following administration of EXAMPLES typical compounds in Such a concentration range, the onset The present invention is described by reference to the of paralysis normally develops within 1 to 2 minutes, and is following Examples, which are offered by way of illustration reversible (i.e., muscle tone returns within a short period of 50 and are not intended to limit the invention in any manner. time). The compounds of this invention would normally be Standard techniques well known in the art or the techniques readministered every 15 to 30 minutes after initial admin specifically described below were utilized. istration or given as a Slow continuous infusion depending upon the length of time a muscular block is desired, and as Example 1 determined by the anesthetist and Surgeon in charge of the 55 patient. For adult human patients undergoing long Surgical Dose-Effect Study for MI and GI procedures, the effective dose of typical compounds is This Study was an open label, dose-ranging, Single center administered through continuous or intermittent intravenous investigation. A total of 14 rats were studied (10 in each of perfusion at a rate from about 0.001 mg/min to about 0.8 five groups). All animals were anesthetized with pentobar mg/min, preferably from about 0.01 mg/min to about 0.5 60 bital (60 mg/kg) given by intraperitoneal administration and mg/min, and more preferably from about 0.01 to about 0.25 maintained with Supplemental doses as determined by physi mg/min. Following administration of typical compounds in ological monitoring variables. A tracheotomy was per the Specified amounts, the onset of paralysis typically devel formed and the rats were ventilated with room air keeping ops within 1 to 2 minutes and persists for the duration of the P near 35 torr. The carotid artery was cannulated to Superfusion. 65 measure blood pressure and arterial blood gases. The right For human patients in the pediatric population undergoing jugular vein was cannulated for intravenous infusion and Short Surgical procedures, the effective dose of typical further drug administration. Body temperature was main US 6,268.473 B1 17 18 tained at 36-38 C. during the entire experiment. The Sciatic nerve was exposed in the popliteal Space and Stimu TABLE 1-continued lated with train-of-four Stimulation using a DigiStim nerve Stimulator. The tivialis anterior muscle contractoin was Comparison of Neuromuscular Blocking Agents measured by attaching the rathind limb to an isometric force Agent Onset Time Recovery (min transducer to record the evoked response. Prior to adminis tration of the Study drug, baseline measurements of blood (mg/kg) (sec) 25% 75% preSSure, heart rate and muscle contraction force were Sux 60 5-7 1O measured for a five-minute period and at five minute inter (1.0) vals for the duration of the study. Org 9847 8O 8 15 (1.5) The initial dose for analysis was based on biologically Rocuronium 8O 40 60 effective doses determined in mice. Based on the onset, (0.6) maximum effect and duration of effect from the first animal Mivacurium 150 2O 27 studied, the dose for the next animal was either doubled or (0.2) 15 Vecuronium 120-18O 40 60 halved. If the relaxation level was maintained at a maximal (0.1) level for greater than 20 minutes from this initial dose, then Cisatracurium 120-18O 45 60-70 the Subsequent dose Studied was doubled. This progression (0.1) continued until the dose that produced near maximal muscle relaxation was found. For doses of these agents which produced less than The conopeptide derivatives MI and GI were studied in maximum levels of neuromuscular block, dose-response the initial Study. For each compound Studied, the onset of plots can be determined to estimate the EDso dose of these muacle relaxation, duration of relaxation and an estimate of agents. In this context, EDso refers to the dose of agent the EDso was determined from evoked force transducer which is expected to produce half of the maximum relax response. Onset of relaxation is defined as the time for the 25 ation level. The data of this initial study (FIG. 3) shows that evoked response to diminish to 5% of pre-drug baseline. In GI is less potent than MI as reflected in the lower EDso value addition, clinical duration, defined as the time from the for MI (-80 ug/kg for MI compared to ~120 ug/kg for GI). administration of drug until the evoked muscle response These results show that C.-conotoxin peptides are biologi returns to 25% of its pre-drug baseline, and recovery time, cally active at the neuromuscular junction producing skeletal muscle paralysis that mimics the repSonse Seen with non defined as tghe time until evoked response returns to 75% of depolarizing neuromuscular blocking agents given during baseline, were also determined. Data were Summarized for anesthesia. The onset and duration of relaxation is rapid and each compound. short which is highly desirable for a number of clinical The onset and recovery results for both MI and GI are reasons. In this regard, with the rapid onset time, short shown in FIGS. 1 and 2, respectively. MI had a shortest duration and no prolongation of drug effect with large doses, onset of 1.46 minutes. The onset time increased with 35 the clinical benefit of the C-conotoxin peptides exceeds the decreasting dose size as is typical for may neuromuscular currently available non-depolarizing neuromuscular block blocking agents. The recovery time to 25% and 75% of ing agents. In addition to their desirable effect profile, the baseline occurred in approximately 8 and 12 minutes, C-conotoxin peptides appear to have no significant cardio respectively. These recovery times were constant for doses vascular effects on administration. Thus, the desirable effect over 100 tug/kg, which implies that recovery of thge drug 40 profile with minimal Side effects are desirable clinical prop effects is very rapid and not easily Saturated in its capacity. erties for the C-conotoxin peptides. Anesthetic drugs that behave in Similar fashion tend to be Example 2 degraded by chemical or enzymatic processes in the body rather than by metabolic organ transformation. 45 Dose-Effect Study for Iodinated-MI GI had a shorter onset time of just under 1 minute. The A Similar study as described in Example 1 was conducted time for 25% and 75% recovery of baseline was in the range for two iodinated derivatives of MI, namely, mono-iodo of 8 and 15 minutes, respectively. AS with MI, increasing the Tyra-MI and di-iodo-Tyr-MI. The onset and recovery dose tended to shorten the onset time without extending the results for mono-iodo-Tyr-MI and di-iodo-Tyr-MI are recovery times dramatically. For GI, the onset time was 50 shown in FIGS. 4 and 5, respectively. Dose-response plots Similar to that seen with Succinylcholine. The recovery times for mono-iodo-Tyr-MI and di-iodo-Tyr-MI were made for both agents were similar to Succinylcholine. to estimate the EDso dose of these agents. The EDso values are ~16 ug/kg for mono-iodo-Tyr-MI and 92.5 lug/kg for A comparison of these results to onset and recovery times di-iodo-Tyr-MI. for other clinically available neuromuscular blocking agents is shown in Table 1. 55 Example 3

TABLE 1. Muscle Relaxant Effect in Anesthetized Monkeys The peptides MI, GI, EI, mono-iodo-MI and di-iodo-MI Comparison of Neuromuscular Blocking Agents are each Separately dissolved 0.9 percent Saline at a con 60 Agent Onset Time Recovery (min centration of 2 mg/ml. Rhesus monkeys are anesthetized with halothane, nitrous oxide and oxygen. The maintenance (mg/kg) (sec) 25% 75% concentration of halothane is 1.0%. Arterial and venous MI 90 8 12 catheters are placed in the femoral vessels for drug admin (0.15) istration and recording of the arterial pressure. Controlled GI 60-70 6-8 10-15 65 ventilation is accomplished via an endotrachael tube. Twitch (0.2) and tetanic contractions of the tibialis arterior muscle are elicited indirectly via the Sciatic nerve. Recordings of arte US 6,268.473 B1 19 20 rial pressure electrocardiogram (lead I), heart rate, and cedures well known in the art, Such as described in Olivera muscle function are made Simultaneously. Four to six ani et al. (1996). Alternatively, cDNA libraries was prepared mals received each listed compound. Four additional ani from Conus Venom duct using conventional techniques. mals received Succinylcholine chloride or d-tubocurarine DNA from single clones was amplified by conventional chloride as controls. IS is seen that the tested compounds techniques using primers which correspond approximately generally provide Similar or better results than those Seen for to the M13 universal priming site and the M13 reverse Succinylcholine chloride or d-tubocurarine chloride. universal priming Site. Clones having a size of approxi mately 300 nucleotides were Sequenced and Screened for Example 4 Similarity in Sequence to known C.-conotoxins. The DNA Sequences and encoded propeptide or peptide Sequences are Isolation of DNA Encoding C.-Conotoxins set forth in Tables 2-26. It was discovered that the following DNA coding for C-conotoxins was isolated and cloned in mature C-conotoxin peptides had the same sequence: (a) accordance with conventional techniques using general pro R1.4, A1.1, Bt1.6, Cn 1.1 and MnI; and (b) Sm1.1 and Cr1.1.

TABLE 2 DNA Sequence (SEQ ID NO: 28) and Protein Sequence (SEQ ID NO : 29) of GI

atg titc. acc gtg titt citg ttg gtg gtc ttg gca acc act gtc gtt to c Met Phe Thr Wall Phe Teu Telu Wall Wall Teu Ala Thr Thr Wall Wall Ser

titc. cct toa gaa cgt. gca tot gat ggC agg gat gac aCa gcc a.a.a. gac Phe Pro Ser Glu Arg Ala Ser Asp Gly Arg Asp Asp Thr Ala Lys Asp

gaa ggg tot gac atg gag a.a.a. ttg gtc gag a.a.a. a.a.a. gaa tgt tgc aat Glu Gly Ser Asp Met Glu Lys Telu Wall Glu Lys Lys Glu Cys Cys Asn

cct gcc tgt ggC aga cac tac agt tgt gga cgc tgatgctoca ggacccitctg Pro Ala Cys Gly Arg His Tyr Ser Cys Gly Arg

aaccacggac gtgc cgc.cct cit gcct gacc tgcttcactg tocgtc.tc.tt tgtgccacta gaactgaa.ca gcto gatcca ctag actacc acgttaccto cgtgttctaa aac tacttgg tittagattgc cititt aattitc tagt catact toctgttatt acgtogtoca aaattgaaac aagaacatga ggggtgtcag citcaaacaaa. atcaggcaat gacaaggaaa atgtc.tc.cga to gatc.cgaa aact gtcacc cgtoacticto ttaaccagtt ttagaactga ttaccactag agctitttgta CCaC atcaaa. to aggtotat gtgtgatgtt tottttgcaa aatttaattit ttgagaaaaa aagctoaaaa tgtgggaagt gcttittgatt ttctgacaac ttgttgatcat gtc.cgtttitc agtg agtcta attgcaacct citgttgttgatt ttctitcaccit gttaa.gcaac gCaaagaggt tgtc cataac caggaaag.ca acagacaaag aaatgcttga gaattitcagg ttatagataa ggta aggaaa aaaaggagag citatgggaaa tgatgaaaac aacagataaa ataaattgaa cagt accitac ttgtttcatg gttgatttitt ttittctotga ataatctotg tggacactaa tggC agtcto to citcacccc acgc.cattag taagcttatt ttittctittct ttatccaaga tttg ctgaac at atttagcc ta9atataga cattgctaca tatataatct gacaataaac tittc. atgggC accalatt

TABLE 3 DNA Sequence (SBQ ID NO:30) and Protein Sequence (SEQ ID NO:31) of SIB

atg titc. acc gtg titt citg ttg gtt gro ttg gca acd act gtc grt to c Met Phe Thr Wall Phe Teu Telu Wall Wall Teu Ala Thr Thr Wall Wall Ser

titc. cct toa gat cgt. gca tot gat ggC agg gat gac gaa gcc a.a.a. gac Phe Pro Ser Asp Arg Ala Ser Asp Gly Arg Asp Asp Glu Ala Lys Asp

gaa agg tot gac atg cac gaa tog gac Cgg a.a.a. gaa atc tgt tgc aat Glu Arg Ser Asp Met His Glu Ser Asp Arg Lys Glu Ile Cys Cys Asn

cct gcc tgt ggC cca aag tat agt tgt gga cgc tgatgctoca ggacccitctg Pro Ala Cys Gly Pro Lys Tyr Ser Cys Gly Arg

alacc

TABLE 4 DNA Sequence (SEQ ID NO:32) and Protein Sequence (SEQ ID NO:31) of R1 atg titc acc gtg titt citg ttg gtt gtc titg aca atc act gtc gtt toc Met Phe Thr Wall Phe Lieu. Leu Wal Wall Leu Thir Ile Thr Wal Wal Ser US 6,268.473 B1 21 22

TABLE 4-continued DNA Sequence (SEQ ID NO:32) and Protein Sequence (SEQ ID NO:31) of R1 titc cct tca gat cqt gca tot gat ggc agg gat gac gaa gCC aaa gac Phe Pro Ser Asp Arg Ala Ser Asp Gly Arg Asp Asp Clu Ala Lys Asp gala agg tot gac atg tac aaa tog aaa cqg aat gga cqc tot togc car Glu Arg Ser Asp Met Tyr Lys Ser Lys Arg Asn Gly Arg Cys Cys His cct gcc togt ggc aaa cac titt agt tot gga cqc to atgct coa ggacccitctg Pro Ala Cys Gly Lys His Phe Ser Cys Cly Arg alaccacgacg t

15

TABLE 5 DNA Sequence (SEQ ID NO:34) and Protein Sequence (SEQ ID NO:35) of R1.3 atg titc acc gtg titt citg ttg gtg gtc ttg gCa acc act gttc gtt to c Met Phe Thr Wall Phe Leu Leu Wal Wall Leu Ala Thir Thr Wal Wal Ser titc cct tca gaa cqt gca tot gat ggc agg gat gac aca gCC aaa gac Phe Pro Ser Glu Arg Ala Ser Asp Gly Arg Asp Asp Thr Ala Lys Asp gala ggg tot gac atg gag aaa ttg gtc gag aaa aaa gaa tot toc aat Glu Cly Ser Asp Met Glu Lys Lieu Val Glu Lys Lys Glu Cys Cys Asn cct gcc togt ggc aga cac tac agt tot aag gga ggacgctgat gcticcagacc Pro Ala Cys Gly Arg His Tyr Ser Cys Lys Gly citctgaacca cqacgt.

TABLE 6 DNA Sequence (SEQ ID NO:36) and Protein Sequence (SEQ ID NO:37) of R1 .. 4 atg titc acc gtg titt citg ttg gtt gtc titg aca atc act gtc gtt to c Met Phe Thr Wall Phe Leu Leu Wal Wall Leu Thir Ile Thr Wal Wal Ser titc cct tca gat cqt gca tot gat ggc agg gat gac gaa gCC aaa gac Phe Pro Ser Asp Arg Ala Ser Asp Gly Arg Asp Asp Glu Ala Lys Asp gala agg tot gac atg tac aaa tog aaa cqg aat gga cqc tot togc cat Glu Arg Ser Asp Met Tyr Lys Ser Lys Arg Asn Cly Arg Cys Cys His cct gcc togt ggc aaa cac titt agt tot gga cqc to atgct coa ggacccitctg Pro Ala Cys Gly Lys His Phe Ser Cys Gly Arg alaccacgacg t

TABLE 7 DNA Sequence (SBQ ID NO: 28) and Protein Sequence (SEQ ID NO:39) of Sml. 1 atg titc acc gtg titt citg ttg gtt gtc titg goa acc act gtc gtt to c Met Phe Thr Wall Phe Leu Leu Wal Wall Leu Ala Thir Thr Wal Wal Ser titc cct tca gat cqt gca tot gat ggc agg gat gac gaa gCC aaa gac Phe Pro Ser Asp Arg Ala Ser Asp Gly Arg Asp Asp Glu Ala Lys Asp gala agg tot gac atg cac gaa to g g g c cqg aaa goa cqc gga cqc tot Glu Arg Ser Asp Met His Glu Ser Gly Arg Lys Gly Arg Gly Arg Cys tgc cat cot goc tot go coa aac tat agt tot goacgctgat gct coaggac Cys His Pro Ala Cys Gly Pro Asn Tyr Ser Cys

US 6,268.473 B1 25 26

TABLE 11-continued DNA Sequence (SEQ ID NO : 46) and Protein Sequence (SEQ ID NO: 47) of GIB citcc gatcga to cqaaaact gtcaccc.gto acticitcttaa. ccagttittag aactgattac cactagagct tttgtaccac atcaaatcag gtotatotgt gatgtttctt ttgcaaaatt taatttittga gaaaaaaagc ticaaaatgtg ggaagtgctt ttgattittct gacaacttgt gatcatgtcc gttittcagtg agtictaattg caaccitctgt gtgattittct to acctgtta agcaac.gcaa. agaggttgtc. catalaccagg aaagcaa.cag acaaagaaat gcttgagaat ttcaggittat agataaggta aggaaaaaaa ggaga.gctat gggaaatgat gaaaacaa.ca gataaaataa attgaacagt accitacttgt ttcatggttg attittitttitt citctgaataa totctgtgga cactaatggc agtctotcct caccc.cacgc cattagtaag cittattittitt citttctititat ccaagatttg citgaacatat ttagoctaga tatagacatt gctacatata taatctgaca ataaacttitc atggg cacca att

15

TABLE 12

DNA Sequence (SEQ ID NO: 48) and Protein Sequence (SEQ ID NO: 49) of MnII atg titc. acc gtg titt citg ttg gtt gtc. ttg aca acc act gtc gtt to c Met Phe Thr Wall Phe Leu Lleu Wall Wall Leu. Thr Thir Thr Wall Wal Ser titc. cct toa gat agt gca tot ggit ggc agg gat gac gag gCC aaa gac Phe Pro Ser Asp Ser Ala Ser Gly Gly Arg Asp Asp Glu Ala Lys Asp gaa agg tot gac arg tac gaa ttg aaa cqg aat gga cac tot tgc cat Glu Arg Ser Asp Met Tyr Glu Lieu Lys Arg Asn Gly His Cys Cys His cct gcc tgt ggit ggC aaa tac gtt aaa tot gga cgc tigatgct cca Pro Ala Cys Gly Gly Lys Tyr Val Arg ggaccotcitc galaccacg

TABLE 13 DNA Sequence (SEQ ID NO: 50) and Protein Sequence (SEQ ID NO: 50) of A1.2 atg titc. acc gtg titt citg ttg gtt gtc. ttg aca acc act gtc gtt to c Met Phe Thr Wall Phe Leu Lleu Wall Wall Leu. Thr Thir Thr Wall Wal Ser titc. cct toa gat agt gca tot ggit ggc agg gat gac gag gCC aaa gac Phe Pro Ser Asp Ser Ala Ser Gly Gly Arg Asp Asp Glu Ala Lys Asp gaa agg tot gac arg tac gaa ttg aaa cqg aat gga cac tot tgc cat Glu Arg Ser Asp Met Tyr Glu Lieu Lys Arg Asn Gly His Cys Cys His cct gcc tgt ggit ggC aaa tac gtt aaa tot gga cgc tigatgct cca Pro Ala Cys Gly Gly Lys Tyr Val Lys Cys Gly Arg ggaccotcitc galaccacg

TAB LE 1.4 DNA Sequence (SEQ ID NO:52) and Protein Sequence (SEQ ID NO:53) of A1. 1 atg titc. acc gtg titt citg ttg gtt gtc. ttg aca aca act gtc gtt to c Met Phe Thr Wall Phe Leu Lleu Wall Wall Leu. Thr Thir Thr Wall Wal Ser tac cct toa gat agt gca tot gat ggc agg gat gac gaa gC c aaa gac Pro Ser Asp Ser Ala Ser Asp Gly Arg Asp Asp Glu Ala Lys Asp gaa agg tot gac atg tac aaa tog aaa cqg aat gga cqc tot tgc cat Glu Arg Ser Asp Met Tyr Lys Ser Lys Arg Asn Gly Arg Cys Cys His US 6,268.473 B1 27 28

TABLE 14-continued DNA Sequence (SEQ ID NO:52) and Protein Sequence (SEQ ID NO:53) of A1. 1 cct gcc togt ggc aaa cac titt agt tot gga cqc to atgct coa ggacccitctg Pro Ala Cys Gly Lys His Phe Ser Cys Gly Arg alaccacgacg t

TABLE 1.5 DNA Sequence (SEQ ID NO :54) and Protein Sequence (SEQ ID NO:55) of Bt 1.6 atg titc acc gtg titt citg ttg gtt gtc titg goa acc act gtc gtt to c Met Phe Thr Wall Phe Leu Leu Wal Wall Leu Ala Thir Thr Wal Wal Ser tac cot to a gat agt gca tot gat ggc agg gat gac gaa acc aaa gac Tyr Pro Ser Asp Ser Ala Ser Asp Gly Arg Asp Asp Glu Thir Lys Asp gala aag tot gac atg tac aaa tog aaa cqg aat gga cqc tot togc cat Glu Lys Ser Asp Met Tyr Lys Ser Lys Arg Asn Gly Arg Cys Cys His cct gcc togt ggc aaa cac titt agt tot gga cqc to atgct goa ggacccitctg Pro Ala Cys Gly Lys His Phe Ser Cys Gly Arg alaccacgacg t

TABLE 16 DNA Sequence (SEQ ID NO:56) and Protein Sequence (SEQ ID NO:57) of Cn 1.1 atg titc acc gtg titt citg ttg gtt gtc titg aca acc act gtc gtt to c Met Phe Thr Wall Phe Leu Leu Wal Wall Leu Thir Thir Thr Wal Wal Ser titc cct tca gat agt gca tot gat gtc agg gat gac gaa gCC aaa gac Phe Pro Ser Asp Ser Ala Ser Asp Val Arg Asp Asp Glu Ala Lys Asp gala agg tot gac atg tac aaa tog aaa cqg aat gga cqc tot togc cat Glu Arg Ser Asp Met Tyr Lys Ser Lys Arg Asn Gly Arg Cys Cys His cct gcc togt ggc aaa cac titt agt tot gga cqc to atgct coa ggacccitctg Pro Ala Cys Gly Lys His Phe Ser Cys Gly Arg alaccacgacg t

TABLE 1.7 DNA Sequence (SEQ ID NO :58) and Protein Sequence (SEQ ID NO: 59) of Mini atg titc acc gtg titt citg ttg gtt gtc titg aca aca act gtc gtt to c Met Phe Thr Wall Phe Leu Leu Wal Wall Leu Thir Thir Thr Wal Wal Ser tac cot to a gat agt gca tot gat ggc agg gat gac gaa gCC aaa gac Tyr Pro Ser Asp Ser Ala Ser Asp Gly Arg Asp Asp Glu Ala Lys Asp gala agg tot gac atg tac aaa tog aaa cqg aat gga cqc tot togc cat Glu Arg Ser Asp Met Tyr Lys Ser Lys Arg Asn Gly Arg Cys Cys His cct gcc togt ggc aaa cac titt agt tot gga cqc to atgct coa ggacccitctg Pro Ala Cys Gly Lys His Phe Ser Cys Gly Arg alaccacgacg t US 6,268.473 B1 29 30

TABLE 1.8 DNA Sequence (SEQ ID NO: 60) and Protein Sequence (SEQ ID NO: 61) of Cr1.1 atg titc acc gtg titt citg ttg gtt gtc titg goa gcc act gtc gtt to c Met Phe Thr Wall Phe Leu Leu Wal Wall Leu Ala Ala Thr Wal Wal Ser titc cct tca gat cqt gca tot gat ggc agg gat gac gaa gCC aaa gac Phe Pro Ser Asp Arg Ala Ser Asp Gly Arg Asp Asp Glu Ala Lys Asp gala aga. tct gac atg cac gaa tog gac cqg aaa goa cqc gga cqc tot Glu Arg Ser Asp Met His Glu Ser Asp Arg Lys Gly Arg Gly Arg Cys tgc cat cot goc tot go coa aat tat agt tot goa cqc to atgctoca Cys His Pro Ala Cys Gly Pro Asn Tyr Ser Cys Gly Arg ggaccotctgaac cacgacg

TABLE 1.9 DNA Sequence (SEQ ID NO : 62) and Protein Sequence (SEQ ID NO: 63) of R1.2 atg titc acc gtg titt citg ttg gtg gtc ttg gCa acc act gttc gtt to c Met Phe Thr Wall Phe Leu Leu Wal Wall Leu Ala Thir Thr Wal Wal Ser titc cct tca gaa cqt gca tot gat ggc agg gat gac aca gCC aaa gac Phe Pro Ser Glu Arg Ala Ser Asp Gly Arg Asp Asp Thr Ala Lys Asp gala ggg tot gac atg gac aaa ttg gtc gag aaa aaa gaa tot togc cat Glu Gly Ser Asp Met Asp Lys Lieu Val Glu Lys Lys Glu Cys Cys His cct gcc togt ggc aaa cac titc agt tot gga cqc to atgct coa ggacccitctg Pro Ala Cys Gly Lys His Phe Ser Cys Gly Arg alaccacgacg t

35

TABLE 2.0 TABLE 21-continued

DNA Sequence (SEQ ID NO : 64) and Protein Sequence 40 DNA Sequenee (SEQ ID NO : 66) and Protein Sequence (SEQ ID NO: 65) of A1.3 (SEQ ID NQ: 67) of A1.7 tot gat ggc agg gat gac gala gCC aaa gac gaa agg Ser Asp Gly Arg Asp Asp Glu Ala Lys Asp Glu Arg tgc cat cot gcc tdt ggc aaa cactitt agt tot gga 45 Cys His Pro Ala Cys Gly Lys His Phe Ser Cys Gly tot gac atg tac aaa tog aaa cqg aat gga cqc tot Ser Asp Met Tyr Lys Ser Lys Arg Asn Gly Arg Cys cgc toga Arg tgc cac cot goc tot go aaa cactitt att tot gga 50 Cys His Pro Ala Cys Gly Lys His Phe Ile Cys Gly TABLE 22 cgc toga DNA Sequence (SEQ ID NO : 68) and Protein Sequence Arg 55 (SEQ ID NO: 69) of A1.8 tot gat ggc agg gat gac gala gCC aaa gac aaa agg Ser Asp Gly Arg Asp Asp Glu Ala Lys Asp Lys Arg TABLE 21 tot gac atg tac gaa tog gac cqg aat gga cqc tot DNA Sequenee (SEQ ID NO : 66) and Protein Sequence 60 Ser Asp Met Tyr Glu Ser Asp Arg Asn Gly Arg Cys (SEQ ID NQ: 67) of A1.7 tgc cat cot toc tot go aga aag tat aat tdt gga tot ggt ggc agg gat gaC gaa gCC aaa gaC gaa agg Cys His Pro Ser Cys Gly Arg Lys Tyr Asn. Cys Gly Ser Gly Gly Arg Asp Asp Glu Ala Lys Asp Glu Arg cgc toga tot gac atg tac gaa tog gac cqg aat gga cqc tot 65 Arg Ser Asp Met Tyr Glu Ser Asp Arg Asn Gly Arg Cys US 6,268.473 B1 31 32

TABLE 23 DNA Sequence (SEQ ID NO : 70) and Protein Sequence (SEQ ID NO: 71) of Ay1.1 totgatggca gggatgacga agccaaagac gaaaggtotg acatgitac gaa tog gac Glu Ser Asp cgg aat gga cqc tot togc cat cot gcc tdt gcg aga aag tat aat tdt Arg Asn Gly Arg Cys Cys His Pro Ala Cys Ala Arg Lys Tyr Asn. Cys gga cqc rgatgctcca ggacccitctgaaccacgacg t Gly Arg

TABLE 24 DNA Sequence (SEQ ID NO : 72) and Protein Sequence (SEQ ID NO: 73) of Ay1. 1 a totgatggca gggatgacga agccaaagac gaaaggtotg acatgitac gaa tog gag Glu Ser Glu cgg aat gaa cqc tot togc cat cot gcc tdt gcg aga aag tat aat tdt Arg Asn. Glu Arg Cys Cys His Pro Ala Cys Ala Arg Lys Tyr Asn. Cys gga cqc to atgctcca ggacccitctgaaccacgacg t Gly Arg

TABLE 25 DNA Sequence (SEQ ID NO : 74) and Protein Sequence (SEQ ID NO:75) of M1.1 atg titc acc gtg titt citg ttg gtt gtc titg aca acc act gtc gtt to c Met Phe Thr Wall Phe Leu Leu Wal Wall Leu Thir Thir Thr Wal Wal Ser titc cct tca gat cqt gca tot gat ggc agg gat gac gaa gCC aaa gac Phe Pro Ser Asp Arg Ala Ser Asp Gly Arg Asp Asp Glu Ala Lys Asp gala agg tot gac atg tac gaa tog aaa cqg gat gga cqc tot togc cat Glu Arg Ser Asp Met Tyr Glu Ser Lys Arg Asp Gly Arg Cys Cys His cct gcc togt ggg caa aac tat agt tot gga cqc to atgct coa ggacccitctg Pro Ala Cys Gly Glin Asn Tyr Ser Cys Gly Arg alaccacgacg t

TABLE 26 DNA Sequence (SEQ ID NO: 76) and Protein Sequence (SEQ ID NO: 77) of M1.3 tot gat ggc agg gat gac gala gCC aaa gac gaa agg cct gac atg tac Ser Asp Gly Arg Asp Asp Glu Ala Lys Asp Glu Arg Pro Asp Met Tyr aaa tog aaa cqg gat gga cqc tot togc cat cot gcc tdt gcg aaa cac Lys Ser Lys Arg Asp Gly Arg Cys Cys His Pro Ala Cys Ala Lys His titt aat tdt gga cqc tatgcticca ggacccitctgaac cacgacg t Phe Asn. Cys Gly Arg US 6,268.473 B1 33 34

TABLE 27 DNA Sequence (SEQ ID NO: 78) and Protein Sequence (SEQ ID NO: 79) of M1 .. 4 tot gar gg.c agg gat gac gala gCC aaa gac gaa agg tot gac atg tac Ser Asp Gly Arg Asp Asp Glu Ala Lys Asp Glu Arg Ser Asp Met Tyr gala tog aaa cqg aat gga cqc tot togc cat cot goc togt gcg aaa aac Glu Ser Lys Arg Asn Gly Arg Cys Cys His Pro Ala Cys Ala Lys Asn tat agt tot gga cqc tatgcticca ggacccitctgaaccacgacg t Tyr Ser Cys Gly Arg

TABLE 28 DNA Sequenee (SEQ ID NO: 80) and Protein Sequence (SEQ ID NO: 81) of M1.5 tot gat ggc agg gat gac gala gCC aaa gac gaa agg tot gac atg tac Ser Asp Gly Arg Asp Asp Glu Ala Lys Asp Glu Arg Ser Asp Met Tyr gala to g g ac cqg aat gga cqc tot togc cat cot gcc tdt gcg aga aag Glu Ser Asp Arg Asn Gly Arg Cys Cys His Pro Ala Cys Ala Arg Lys tat aat tdt gga cqc tatgcticca ggacccitctgaac cacgacg t Tyr Asn. Cys Gly Arg

TABLE 29 DNA Sequence (SEQ ID NO: 82) and Protein Sequence (SEQ ID NO: 83) of O1.3 totgatggca gggatgacac agccaaaaac aaaggatctg acatgaacaa attg gtc Wall aag aaa aaa caa tot togc aat cot gcc tdt ggc cca aag tat agt tot Lys Lys Lys Glin Cys Cys Asn Pro Ala Cys Gly Pro Lys Tyr Ser Cys gga cac to atgctcca ggacccitctgaaccacgacg t Gly His

TABLE 30 DNA Sequence (SEQ ID NO: 84) and Protein Sequence (SEQ ID NO: 85) of S1.3 tot gat ggc agg gat gac gala gCC aaa gac gaa agg tot gac at g cac Ser Asp Gly Arg Asp Asp Glu Ala Lys Asp Glu Arg Ser Asp Mer His gala to g g ac cqg aaa goa cqc goa tac tot togc cat cot goc tot ggc Glu Ser Asp Arg Lys Gly Arg Ala Tyr Cys Cys His Pro Ala Cys Gly aaa aag tat aat tdt gga cqc to atgctoca ggacccitctgaac cacgacg t Lys Lys Tyr Asn. Cys Gly Arg

55 TABLE 31 TABLE 31-continued DNA Sequence (SEQ ID NO: 86) and Protein Sequence DNA Sequence (SEQ ID NO: 86) and Protein Sequence (SEQ ID NO: 87) of EI (SEQ ID NO: 87) of EI atg titc acc gtg titt citg ttg gtt gtc ttg gCa acc 60 Gly Arg Asp Ala Ala Ala Asn Asp Lys Ala Ser Asp Met Phe Thr Wall Phe Leu Leu Wal Wall Leu Ala Thr citg atc gct citg acc gcc agg aga gat coa toc tot act gtc. g.gt toc titc act tta gat cqt gca tot gat Lieu. Ile Ala Lieu. Thr Ala Arg Arg Asp Pro Cys Cys Thr Val Gly Ser Phe Thr Leu Asp Arg Ala Ser Asp 65 tac cat cot acc tdt aac atg agit aat coa cag att ggit agg gat gcc gca gcc aac gac aaa gC g tot gac Tyr His Pro Thr Cys Asn Met Ser Asn Pro Glin Ile US 6,268.473 B1 35 36

TABLE 31-continued TABLE 34. DNA Sequence (SEQ ID NO: 86) and Protein Sequence (SEQ ID NO: 87) of EI 5 DNA Sequence (SEQ ID NO :92) and Protein Sequence (SEQ ID NO: 93) of P1.3 tgt ggit Cys Gly atg titc acc gtg titt citg ttg gtt gtc titg gta acc tgaagacgct gatgcticcag gacccitctga accacgacgt. Met Phe Thr Wall Phe Leu Leu Wal Wall Leu Wall Thr 10 acc gtc gtt toc titc aat to a gat cqt gca tta ggit TABLE 32 Thr Val Val Ser Phe Asn Ser Asp Arg Ala Leu Gly DNA Sequence (SEQ ID NO: 88) and Protein Sequence (SEQ ID NO: 89) of EIA 15 ggc agg aat gct gca gcc aaa gC g tot gac aag atc atg titc acc gtg titt citg ttg gtt gtc ttg gCa acc Gly Arg Asn Ala Ala Ala Lys Ala Ser Asp tys Ile Met Phe Thr Wall Phe Leu Leu Wal Wall Leu Ala Thr act gtc. g.gt toc titc act tta gat cqt gca tot gat gct tcg atc citc ggg aga aga gCa toc tot tot tat Thr Val Gly Ser Phe Thr Leu Asp Arg Ala Ser Asp 20 Ala Ser Ile Leu Gly Arg Arg Ala Cys Cys Ser Tyr ggit agg gat gcc gca gcc aac gac aaa gC g tot gac Gly Arg Asp Ala Ala Ala Asn Asp Lys Ala Ser Asp cct coc togt aac gtgaac tat coa gaa att tot got citg atc gct citg acc gcc agg aga gat coa toc tot Pro Pro Cys Asn Val Asn Tyr Pro Glu Ile Cys Gly Lieu. Ile Ala Lieu. Thr Ala Arg Arg Asp Pro Cys Cys 25 toc aat cot goc tdt aac gitg aat aat coa cag att gga cqa ggc Ser Asn Pro Ala Cys Asn. Wall Asn. Asn Pro Glin Ile Gly Arg Gly tgt ggit Cys Gly tgatgctcca ggaccctcrg aaccacgacg t tgaagacgct gatgcticcag gacccitctga accacgacgt.

TABLE 33 DNA Sequence (SEQ ID NO: 90) and Protein Sequence (SEQ ID NO: 91) of P1.2 atg titc acc gtg titt citg ttg gtg gat gcc goa gcc aac gac aag gC g Met Phe Thr Val Phe Lieu Lleu Val Asp Ala Ala Ala Asn Asp Lys Ala tot gac cqg atc gct citg acc goc agg aga gat coa toc tot to c aat Ser Asp Arg Ile Ala Lieu. Thir Ala Arg Arg Asp Pro Cys Cys Ser Asn cct gtc. tot acc gtg cat aat coa cag att tot got togalagacgct Pro Val Cys Thr Val His Asn Pra Glin Ile Cys Gly gatgcticcag gacccitctga accacgacgt. US 6,268.473 B1 37 38

TABLE 35 DNA Sequence (SEQ ID NO: 94) and Protein Sequence (SEQ ID NO: 95) of Sl1.4 atg titc acc gtg titt citg ttg gtt gtc titg goa acc acc gtc gtt coc Met Phe Thr Wall Phe Leu Leu Wal Wall Leu Ala Thir Thr Wal Wall Pro ttcaar to a gat cqt gat coa gCa tta ggt ggc agg aat got gca gCC Phe Asn. Ser Asp Arg Asp Pro Ala Leu Gly Gly Arg Asn Ala Ala Ala ata gC g tot gac aag atc gct tcg acc ctic agg aga gga gga toc tot Ile Ala Ser Asp Lys Ile Ala Ser Thr Lieu Arg Arg Gly Gly Cys Cys tot tat cot coc tot aac gtg to c tat coa gaa att tot ggt gga cqa Ser Tyr Pro Pro Cys Asn Val Ser Tyr Pro Glu Ile Cys Gly Gly Arg cgc to atgctoca gg accotctgaaccacgacgt.

2O TABLE 36 TABLE 38

DNA Sequence (SEQ ID NO: 96) and Protein Sequence DNA Sequence (SEQ ID NO : 100) and Protein Sequence (SEQ ID NO: 97) of Sl1. 4A (SEQ ID NO : 101) of P1.1 25 atg titc acc gtg titt citg ttg gtt gtc ttg gca acc atg titc acc gtg titt citg ttg gtt gtc ttg gCa acc Met Phe Thr Wall Phe Leu Leu Wal Wall Leu Ala Thr Met Phe Thr Wall Phe Leu Leu Wall, Wall Leu Ala Thr act gtc. g.gt toc titc act tta gat cqt gca tot gat acc gtc gtt toc titc aat tda gat cqt gca tta ggit Thr Wallwa Gwy Sser Phee Thrr LLieu. A.Asp A.Arg Alaa Sser A.Asp Thr Val Val Ser Phe Asn Ser Asp Arg Ala Leu Gly ggit agg gar gcc gca gcc aac gac aaa gCd act gac 3O Gly Arg Asp Ala Ala Ala Asn Asp Lys Ala Thr Asp ggc agg aat gct gca gcc aaa gC g tot gac aag atc Gly Arg Asn Ala Ala Ala Lys Ala Ser Asp Lys Ile citg atc gct citg acc gcc agg aga gat coa toc tot Lieu. Ile Ala Lieu. Thr Ala Arg Arg Asp Pro Cys Cys gct tcg atc ctic ggg aga aga aga toc tot tot tat to c aat cor gtc tdt acc gtg cat aat coa cag att Ala Ser Ile Leu Gly Arg Arg Arg Cys Cys Ser Tyr 35 Ser Asin Pro Val Cys Thr Val His Asn Pro Glin Ile tgt ggit cct coc togt aac gitg toc tat coa gaa att togt ggit Cys Gly Pro Pro Cys Asn Val Ser Tyr Pro Glu Ile Cys Gly tgaagacgct gatgcttcag gacccitctga accacgacgt. gga cqa cqC 40 Gly Arg Arg It will be appreciated that the methods and compositions of the instant invention can be incorporated in the form of a tgatgctoca ggacccitctgaac cacgacg t variety of embodiments, only a few of which are disclosed herein. It will be apparent to the artisan that other embodi ments exist and do not depart from the spirit of the invention. Thus, the described embodiments are illustrative and should TABLE 37 not be construed as restrictive. DNA Sequence (SEQ ID NO: 98) and Protein Sequence LIST OF REFERENCES (SEQ ID NO: 99) of Sl1.8 - 50 Blount, K. et al. (1992). Toxicon 30:835-842. atg titc acc gtg titt citg ttg gtt gtc ttg gCa acc Bodansky et al. (1966). Chem. Ind. 38:1597–98. Met Phe Thr Wall Phe Leu Leu Wal Wall Leu Ala Thr Cruz, L. J. et al. (1987). J. Biol. Chem. 260:9280–9288. acc gtc gtt toc titc aat tda gat cqt gca tta ggit Goodman and Gilman's The Pharmacological Basis of Thr Val Val Ser Phe Asn Ser Asp Arg Ala Leu Gly Therapeutics, 7th Ed., Section II (1985). it cost tot t 55 Gray, W. R. et al. (1981). J. Biol. Chem. 256:4734–4740. Glyggc Argagg Asnaat Alagct Alagca Alagcc aaaLys AlagC g totSer Aspgac aagLys atcIle Haack, J. A. et al. (1990).J. Biol. Chen. 265:6025-6029. Horiki, K. et al. (1978). Chemistry Letters 165–68. gct tcg atc ctic ggg aga aga gCa toc tot tot tat Kapoor (1970). J. Pharm. Sci. 59:1-27. Ala Ser Ile Leu Gly Arg Arg Ala Cys Cys Ser Tyr Kornreich, W. D. et al. (1986). U.S. Pat. No. 4,569,967. cct coc togt aac gtgaac tat coa gaa att tot got 60 Marshall, I. G. and Harvey, A. L. (1990). Toxicon Pro Pro Cys Asn Val Asn Tyr Pro Glu Ile Cys Gly 28:231-234. McIntosh, J. M. et al. (1982). Arch. Biochem. BiophyS. gga cqa ggc 218:329-334. Gly Arg Gly Mena, E. E. et al. (1990). Neurosci. Lett. 118:241-244. tgatgctoca ggacccitctgaac cacgacg t 65 Methoden der Organischen Chemie (Houben-Weyl): Syn these von Peptiden, E. Wunsch (Ed.), Georg Thieme Verlag, Stuttgart, Ger. (1974). US 6,268.473 B1 39 40 Myers, R. A. et al. (1991). Biochemistry 30:9370-9377. ASn-Gly-Arg-CyS-Cys-His-Xaa-Ala-Cys-Ala-Arg Nishiuchi, Y. et al. (1993). Int. J. Pept. Protein Res. Xaa-Xaa-Ser-Cys (SEQ ID NO:18); 42:533-538. Xaas-CyS-CyS-ASn-Xaa-Ala-CyS-Gly-Xaa-Xaa-Xaa Nowak, L. et al. (1984). Nature 307:462-465. Ser-Cys (SEQ ID NO:19); Olivera, B. M. et al. (1984). U.S. Pat. No. 4,447,356. Xaas-CyS-Cys-His-Xaa-Ala-CyS-Gly-Xaa-Xaa-Xaa Olivera, B. M. et al. (1985). Science 230:1338–1343. ASn-Cys (SEQ ID NO:20); Olivera, B. M. et al. (1996). U.S. Pat. No. 5,514,774. Ser-Gly-Arg-CyS-Cys-His-Xaa-Ala-CyS-Gly-Arg-Xaa Physicians' Desk Reference, 48th Ed., pp. 689,758,1362, Xaa-Asn-Cys (SEQ ID NO:21); 1648 (1994). Arg-Asp-Xaa-CyS-CyS-Ser-Asn-Xaa-Val-Cys-Thr-Val Rivier, J. R. et al. (1978). Biopolymers 17:1927-38. 1O His-Asn-Xaa-Gln-Ile-Cys (SEQ ID NO:22); Rivier, J. R. et al. (1987). Biochem. 26:8508-8512. Arg-Ala-CyS-CyS-Ser-Xaa-Xaa-Xaa-CyS-ASn-Val Sambrook, J. et al. (1989). Molecular Cloning: A Labora Asn-Xaa-Xaa-Xaa-Ile-Cys (SEQ ID NO:23); tory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Gly-Gly-Cys-CyS-Ser-Xaa-Xaa-Xaa-CyS-ASn-Val Cold Spring Harbor, N.Y. Ser-Xaa-Xaa-Xaa-Ile-Cys (SEQ ID NO:24); Schroder & Lubke (1965). The Peptides 1:72–75, Academic 15 Ser-Leu-Leu-Cys-Cys-Thr-Ile-Xaa-Ser-Cys-Xaa-Ala Press, NY. Ser-Xaa-Xaa-Asp-Ile-Cys (SEQ ID NO:27), Stewart and Young, Solid-Phase Peptide Synthesis, Freeman wherein Xaa is Glu or Y-carboxy-glutamate (Gla); Xaa & Co., San Francisco, Calif. (1969). is Pro or hydroxy-Pro; Xaa is Tyr, mono-halo-Tyr, Vale et al. (1978). U.S. Pat. No. 4,105,603. di-halo-Tyr, O-sulpho-Tyr, O-phospho-Tyr or nitro Zafaralla, G. C. et al. (1988). Biochemistry 27:7102-7105. Tyr; Xaa, is Lys, N-methyl-LyS, N,N-dimethyl-Lys or Zhou L. M., et al. (1996). J. Neurochem. 66:620–628. N.N.N-trimethyl-Lys, Xaas is Gln or pyro-Glu; and the U.S. Pat. No. 3,972,859. C-terminus contains a carboxyl or amide group, the U.S. Pat. No. 3,842,067. polypeptide being Substantially free of other peptides. U.S. Pat. No. 3,862,925. U.S. Pat. No. 4,190,674. 2. The Substantially pure C-conotoxin peptide of claim 1, U.S. Pat. No. 4,179,507. 25 wherein Xaa is Glu. U.S. Pat. No. 4,508,715. 3. The Substantially pure C-conotoxin peptide of claim 1, U.S. Pat. No. 4,701,460. wherein Xaa is LyS. U.S. Pat. No. 4,761,418. 4. The Substantially pure C-conotoxin peptide of claim 1, U.S. Pat. No. 4,923,898. wherein Xaa is Tyr. U.S. Pat. No. 5,015,741. 5. The Substantially pure C-conotoxin peptide of claim 1, U.S. Pat. No. 5,260,337. wherein Xaa is mono-iodo-Tyr. What is claimed is: 6. The Substantially pure C-conotoxin peptide of claim 1, 1. A Substantially pure C-conotoxin peptide Seleceted wherein Xaa is di-iodo-Tyr. from the group consisting of: 7. The Substantially pure C-conotoxin peptide of claim 1, 35 wherein Xaas is Gln. Xaa-CyS-Cys-Asn-Xaa-Ala-CyS-Gly-Arg-His-Xaa 8. The Substantially pure C-conotoxin peptide of claim 1, Ser-Cys-Xaa-Gly (SEQ ID NO:3); which is modified to contain an O-glycan, an S-glycan or an ASn-Gly-Arg-CyS-Cys-His-Xaa-Ala-CyS-Gly-Xaa N-glycan. His-Phe-Ser-Cys (SEQ ID NO:4); 9. The substantially pure C-conotoxin peptide of claim 1, Gly-Arg-Gly-Arg-CyS-Cys-His-Xaa-Ala-CyS-Gly 40 wherein Said peptide has the amino acid Sequence Set forth Xaa-Asn-Xaa-Ser-Cys (SEQ ID NO:5); in SEQID NO:3 and wherein Xaa is Glu, Xaa- is Pro, Xaa, CyS-Cys-His-Xaa-Ala-Cys-Gly-Arg-Xaa-Xaa-ASn is Tyr and Xaa, is Lys. Cys (SEQ ID NO:6); 10. The substantially pure C-conotoxin peptide of claim 1, CyS-CyS-CyS-ASn-Xaa-Ala-CyS-Gly-Xaa-ASn-Xaa wherein Said peptide has the amino acid Sequence Set forth Gly-Cys-Gly-Thr-Ser-Cys-Ser-Arg-Xaa-Ser-Xaa 45 in SEQ ID NO.4 and wherein Xaa- is Pro and Xaa, is Lys. Xaa-Arg-Arg (SEQ ID NO:7); 11. The Substantially pure C-conotoxin peptide of claim 1, ASn-Gly-His-Cys-Cys-His-Xaa-Ala-Cys-Gly-Gly-Xaa wherein Said peptide has the amino acid Sequence Set forth Xaa-Val-Xaa-Cys (SEQ ID NO:8); in SEQ ID NO:5 and wherein Xaa is Pro and Xaa is Tyr. Asn-Gly-Arg-Cys-Cys-His-Xaa-Ala-Cys-Gly-Gly 12. The Substantially pure C-conotoxin peptide of claim 1, Xaa-Xaa-Val-Xaa-Cys (SEQ ID NO:9); 50 wherein Said peptide has the amino acid Sequence Set forth ASn-Gly-Arg-CyS-Cys-His-Xaa-Ala-CyS-Gly-Xaa in SEQ ID NO:6 and wherein Xaa- is Pro, Xaa is Tyr and His-Phe-Ile-Cys (SEQ ID NO:10); Xaa is LyS. ASn-Gly-Arg-CyS-Cys-His-Xaa-Ala-Cys-Gly-Xaa 13. The Substantially pure C-conotoxin peptide of claim 1, His-Phe-Ser-Cys (SEQ ID NO:11); wherein Said peptide has the amino acid Sequence Set forth 55 in SEQ ID NO:7 and wherein Xaa is Glu, Xaa is Pro and ASn-Gly-Arg-CyS-Cys-His-Xaa-Ser-CyS-Gly-Arg Xaa- is Tyr. Xaa-Xaa-Asn-Cys (SEQ ID NO:12); 14. The Substantially pure C-conotoxin peptide of claim 1, ASn-Gly-Arg-CyS-Cys-His-Xaa-Ala-Cys-Ala-Arg wherein Said peptide has the amino acid Sequence Set forth Xaa-Xaa-Asn-Cys (SEQ ID NO:13); in SEQ ID NO:8 and wherein Xaa- is Pro, Xaa is Tyr and ASn-Xaa-Arg-CyS-Cys-His-Xaa-Ala-Cys-Ala-Arg 60 Xaa is LyS. Xaa-Xaa-Asn-Cys (SEQ ID NO:14); 15. The substantially pure C-conotoxin peptide of claim 1, Asp-Gly-Arg-CyS-Cys-His-Xaa-Ala-CyS-Gly-Gln-ASn wherein Said peptide has the amino acid Sequence Set forth Xaa-Ser-Cys (SEQ ID NO:15); in SEQ ID NO:9 and wherein Xaa is Pro, Xaa, is Tyr and Asp-Gly-Arg-CyS-Cys-His-Xaa-Ala-Cys-Ala-Xaa-His Xaa is LyS. Phe-Asn-Cys (SEQ ID NO:16); 65 16. The Substantially pure C-conotoxin peptide of claim 1, ASn-Gly-Arg-CyS-Cys-His-Xaa-Ala-Cys-Ala-Xaa wherein Said peptide has the amino acid Sequence Set forth Asn-Xaa-Ser-Cys (SEQ ID NO:17); in SEQ ID NO:10 and wherein Xaa- is Pro and Xaa is Lys. US 6,268.473 B1 41 42 17. The substantially pure C-conotoxin peptide of claim 1, 26. The Substantially pure C-conotoxin peptide of claim 1, wherein Said peptide has the amino acid Sequence Set forth wherein Said peptide has the amino acid Sequence Set forth in SEQ ID NO:11 and wherein Xaa is Pro and Xaa is Lys. in SEQ ID NO:20 and wherein Xaa is Pro, Xaa is Tyr, 18. The substantially pure C-conotoxin peptide of claim 1, Xaa is Lys and Xaas is Gln. wherein Said peptide has the amino acid Sequence Set forth 27. The Substantially pure C-conotoxin peptide of claim 1, in SEQ ID NO:12 and wherein Xaa- is Pro and Xaa is Lys. wherein Said peptide has the amino acid Sequence Set forth 19. The substantially pure C-conotoxin peptide of claim 1, in SEQ ID NO:21 and wherein Xaa is Pro, Xaa is Tyr and wherein Said peptide has the amino acid Sequence Set forth Xaa is LyS. in SEQID NO:13 and wherein Xaa is Pro, Xaa is Tyr and 28. The Substantially pure C-conotoxin peptide of claim 1, Xaa is LyS. 1O wherein Said peptide has the amino acid Sequence Set forth 20. The substantially pure C-conotoxin peptide of claim 1, in SEQ ID NO:22 and wherein Xaa is Pro. wherein Said peptide has the amino acid Sequence Set forth 29. The substantially pure C-conotoxin peptide of claim 1, in SEQ ID NO:14 and wherein Xaa is Glu, Xaa- is Pro, wherein Said peptide has the amino acid Sequence Set forth Xaa- is Tyr and Xaa, is Lys. in SEQID NO:23 and wherein Xaa is Glu, Xaa is Pro and 21. The Substantially pure C-conotoxin peptide of claim 1, 15 Xaa- is Tyr. wherein Said peptide has the amino acid Sequence Set forth 30. The substantially pure C-conotoxin peptide of claim 1, in SEQ ID NO:15 and wherein Xaa- is Pro and Xaa is Tyr. wherein Said peptide has the amino acid Sequence Set forth 22. The Substantially pure C-conotoxin peptide of claim 1, in SEQID NO:24 and wherein Xaa is Glu, Xaa- is Pro and wherein Said peptide has the amino acid Sequence Set forth Xaa- is Tyr. in SEQ ID NO:16 and wherein Xaa- is Pro and Xaa is Lys. 31. The Substantially pure C-conotoxin peptide of claim 1, 23. The Substantially pure C-conotoxin peptide of claim 1, wherein Said peptide has the amino acid Sequence Set forth wherein Said peptide has the amino acid Sequence Set forth in SEQID NO:25 and wherein Xaa is Glu, Xaa- is Pro and in SEQID NO:17 and wherein Xaa is Pro, Xaa is Tyr and Xaa- is Tyr. Xaa is LyS. 32. The Substantially pure C-conotoxin peptide of claim 1, 24. The Substantially pure C-conotoxin peptide of claim 1, 25 wherein Said peptide has the amino acid Sequence Set forth wherein Said peptide has the amino acid Sequence Set forth in SEQ ID NO:27 and wherein Xaa- is Pro, Xaa is Tyr and in SEQID NO:18 and wherein Xaa- is Pro, Xaa is Tyr and Xaa is LyS. Xaa is Lys. 33. A Substantially pure C-conotoxin protein precursor 25. The Substantially pure C-conotoxin peptide of claim 1, comprising an amino acid Sequence Selected from the group wherein Said peptide has the amino acid Sequence Set forth of amino acid sequences set forth in Tables 2-38. in SEQ ID NO:19 and wherein Xaa is Pro, Xaa is Tyr, Xaa is LyS and Xaas is Gln. k k k k k

UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTION

PATENT NO. : 6,268.473 B1 Page 2 of 2 DATED : July 31, 2001 INVENTOR(S) : Baldomero M. Olivera et al.

It is certified that error appears in the above-identified patent and that said Letters Patent is hereby corrected as shown below:

Arg-Asp-Xaa-Cys-Cys-Ser-Asn-Xaa-Val-Cys-Thr-Val-His-Asn-Xaa-Gln-Ile-Cys (SEQ ID NO:22). Arg-Ala-Cys-Cys-Ser-Xaa-Xaa-Xaa-Cys-Asn-Val-Asn-Xaa-Xaa-Xaa1-Ile-Cys (SEQID NO:23): Gly-Gly-Cys-Cys-Ser-Xaa-Xaa-Xaa-Cys-Asn-Val-Ser-Xaa-Xaa-Xaa-Ile-Cys (SEQID NO:24); Cys-Cys-Ser-Xaa-Xaa-Xaa-Cys-Asn-Val-Ser-Xaa-Xaa-Xaa-Ile-Cys (SEQID NO:25), Ser-Leu-Leu-Cys-Cys-Thr-Ile-Xaa-Ser-Cys-Xaa-Ala-Ser-Xaa-Xaa-Asp-Ile-Cys (SEQ ID NO:27), wherein Xaa, is Glu or y-carboxy-glutamate (Gla); Xaa, is Pro or hydroxy-Pro; Xaa, is Tyr, mono halo-Tyr, di-halo-Tyr, O-sulpho-Tyr, O-phospho-Tyr or nitro-Tyr; Xaa, is LyS, N-methyl-Lys, N,N- dimethyl-Lys or N,N,N-trimethyl-Lys; Xaa, is Gln or pyro-Glu; and the C-terminus Contains a carboxyl or amide group, the polypeptide being Substantially free of other peptides.

Signed and Sealed this Twenty-third Day of March, 2004 WDJ

JON W. DUDAS Acting Director of the United States Patent and Trademark Office