US007273850B2

(12) United States Patent (10) Patent N0.: US 7,273,850 B2 Stewart et al. (45) Date of Patent: Sep. 25, 2007

(54) PARALYTIC PEPTIDE FOR USE IN Ellis, S., et al. “Properties of a Toxin From the Salivary Gland of the NEUROMUSCULAR THERAPY XShrew, B/arina brevicauda”; The Journal of Pharmacology & Experimental Therapeutics; vol. 114, No. 2, pp. 127-137 (1955). (75) Inventors: John M. Stewart, Sackville (CA); GenCore version 5.1.7, pp. 3-4 (Result 5). Bradley J. Steeves, Woodstock (CA); George, S., et al. “B/arina brevicauda”; Mammalian Species, No. 261, pp. 1-9, 3 ?gs (1986). Karl Vernes, Arrnidale (AU) Kita, M., et al. “Blarina toxin, a mammalian lethal venom from the short-tailed shrew B/arina vrevicauda: Isolation and characteriza (73) Assignee: Bioprospecting NB Inc., New tion.” PNAS, vol. 101, No. 20, pp. 7542-7547 (2004). Brusnwick (CA) Lecchi, P., et al. “The Structure of Synenkephalin (Pro-Enkephalinl_ 73) Is Dictated by Three Disul?de Bridges”; Biochemical and ( * ) Notice: Subject to any disclaimer, the term of this Biophysical Research Communications, vol. 232, No. 3, pp. 800 patent is extended or adjusted under 35 805 (1997). U.S.C. 154(b) by 0 days. Martin, I. “Venom of the Short-Tailed Shrew (B/arina brevicauda) as an Insect ImmobiliZing Agent”; Journal of Mammalogy, vol. 62, (21) Appl. No.: 11/507,128 No. 1, pp. 189-192 (1978). Montell, C. “The Venerable Inveterate Invertebrate TRP Channels”; (22) Filed: Aug. 21, 2006 Cell Calcium 33, pp. 409-417 ((2003). Mount Allison University “Potent Peptide Paralytic Agent”, Version (65) Prior Publication Data 1 (Jun. 2003). Mount Allison University “Potent Peptide Paralytic Agent” Version US 2007/0020251 A1 Jan. 25, 2007 2 (Jul. 2003). Peng, J -B, et al. “CaTl Expression Correlates with Tumor Grade in Related US. Application Data Prostate Cancer”; Biochemical and Biophysical Research Commu nication, vol. 282, pp. 729-734 (2001). (63) Continuation of application No. 10/ 858,233, ?led on Peng, J-B. et al. “Human Calcium Transport Protein CaTl”; Bio Jun. 1, 2004, now Pat. No. 7,119,168, which is a chemical and Biophysical Research Communications, vol. 278, No. continuation-in-part of application No. 10/716,314, 2, pp. 326-332 (2000). ?led on Nov. 18, 2003. Phol, M., et al. “Molecular Cloning of the Helodermin and Exendin-4 cDNAs in the Lizard”; The Journal of Biological Chem (60) Provisional application No. 60/427,682, ?led on Nov. istry, vol. 273, No. 16, pp. 9778-9784 (1998). 18, 2002. Pucek, M. “Chemistry and Pharmacology of Insectivore Venoms”; Chapter 3 of Venomous Animals and Their Venoms edited by W. (51) Int. Cl. Bucher, Academic Press, new York-London, pp. 43-50 (1968). A61K 38/00 (2006.01) Smart, P. “Shrew Saliva Spells Relief? Prof. Jack Stewart makes (52) US. Cl...... 514/12 breakthrough medical discovery”; The Argosy (Jan. 16, 2003). “The venom of the shrew may be in the new Botox”; National Post, (58) Field of Classi?cation Search ...... None Science Section (Biochemistry) (Dec. 20, 2002). See application ?le for complete search history. Tomasi, T. “Function of Venom in the Short-Tailed Shrew B/arina (56) References Cited brevicauda”; Journal of Mammalogy, vol. 59, No. 4, pp. 852-854 (1978). U.S. PATENT DOCUMENTS Zhuang, L., et al. “Calcium-Selective Ion Channel, CaTl, Is Api cally Localized in Gastrointestinal Tract Epithelia and is Aberrantly 5,424,286 A 6/1995 Eng Expressed in Human Malignacies”; Laboratory Investigation, vol. 7,119,168 B2* 10/2006 Stewart et al...... 530/350 82, No. 12, pp. 1755-1764 (2002). FOREIGN PATENT DOCUMENTS * cited by examiner JP 10-236963 9/1998 Primary ExamineriKaren Cochrane Carlson Assistant ExamineriAgnes B. Rooke OTHER PUBLICATIONS (74) Attorney, Agent, or F irmiSynnestvedt & Lechner LLP Christenbury, P. “A Study of the Ecology of B/arina brevicauda in North Carolina and of the Effect of Shrew Toxin on the Liver and (57) ABSTRACT Kidneys of Mice”; A thesis submitted to the Graduate Faculty of Wake Forest College in partial ful?llment of the requirements for The invention relates to a low molecular weight peptide (or the degree of Master of Arts in the Department of Biology; (Aug. suite of related peptides) isolated from the submaxiliary 1966). saliva glands of shrews of the species Blarina as a paralytic Dekker, E., et al. “The epithelial calcium channels, TRPVS and agent. This novel paralytic agent is useful as a neuromus TRPV6: from identi?cation towards regulation”, Cell Calcium 33, cular blocker and analgesic or as an insecticide. pp. 497-507 (2003). Dufton, M. “Venomous Mammals”; Pharmac. Ther. vol. 53, pp. 199-215 (1992). 18 Claims, 16 Drawing Sheets U.S. Patent Sep. 25, 2007 Sheet 1 0f 16 US 7,273,850 B2

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Treatment US 7,273,850 B2 1 2 PARALYTIC PEPTIDE FOR USE IN variant shoWn in FIG. 1B (SEQ ID N012) and other deriva NEUROMUSCULAR THERAPY tives described herein. The inventors further shoW that, While a high molecular Weight fraction is paralytic, the CROSS REFERENCE TO RELATED active molecule is not a large protein but, unexpectedly, a APPLICATION small peptide bound in a large complex of many proteins (FIG. 3, Lane 1). The invention relates to a loW molecular This application is a continuation of US. application Ser. Weight peptide (or optionally a suite of related peptides), No. 10/858,233 ?led on Jun. 1, 2004 now US. Pat. No. preferably, isolated and puri?ed from the submaxiliary 7,119,168, Which is a continuation-in-part of US. applica saliva glands (eg. submaxillary gland) or saliva of shreWs of tion Ser. No. 10/716,314 ?led on Nov. 18, 2003, Which a species such as Blarina as a paralytic agent. The peptide claims priority from US. application No. 60/427,682, ?led optionally has a molecular Weight of about 6000 Da as Nov. 18, 2002, all of Which are incorporated by reference herein in their entirety. measured by SDDS-PAGE. The peptide optionally includes at least one or tWo cysteine amino acids having a sul?hydryl FIELD OF THE INVENTION group and forming a disulfhydryl bond. Optionally, the peptide comprises six cysteine amino acids each having a The invention relates to a paralytic peptide for neuromus sulfhydryl group forming three disulfhydryl bonds. The cular therapy and other uses requiring disruption of neuro peptide optionally absorbs light at 280 nm and more strongly muscular mechanisms. at 260 nm, and includes at least one aromatic amino acid. All 20 or part of the peptide or it parent pro-peptide may also be BACKGROUND OF THE INVENTION produced by recombinant DNA methods or in vitro or in vivo peptide synthesis. This novel paralytic agent is useful ShreWs are a very ancient group of primitive mammals as a neuromuscular blocker. that resemble most closely the proto-mammals. They are not As mentioned above, the active ingredient is a small closely related to rodents because rodents evolved from 25 peptide isolated in an unusual and unexpected combination different groups of mammals. According to Dufton (1992), Within a large protein complex (or a large protein). The the knoWn venomous species of shreW are: the northern peptide is optionally is hydrolytically cleaved from the short-tailed shreW (Blarina brevicauda), the Haitian soleno protein or complex. Known mammalian saliva peptides (e.g. don (Solenodon paradoxus), the European Water shreW vasoactive intestinal polypeptide & glucagon-like peptidel (Neomys fodiens) and the Mediterranean shreW (Neomys 30 [Pohl & Wank 1998]) Would not be contaminants as they are anomalous). Another venomous shreW is the southern short discarded With inactive, loW molecular Weight molecules tailed shrew (Blarina carolinensis). It has also been sug during the puri?cation protocols. The preparation of the gested that the Cuban solenodon (Apologale cubanus), the invention is of great purity and can be extracted from an American shreW (Sorex cinereus) and the Maritime shreW unexpected sub-cellular source. (Sorex marilimensis) could be venomous. The northern 35 short-tailed shreW (Blarina brevicauda) and its closely The present inventors have isolated and puri?ed novel related species use a paralytic venom in its saliva to paralyZe proteins from the submaxilary saliva glands of shreWs. In insects, other invertebrates (Worms, annelids etc.), nesting accordance With one embodiment of the invention, there is birds and small mammals Which it then stores, alive in its provided an isolated and puri?ed shreW saliva peptide. In a speci?c embodiment, the isolated and puri?ed shreW saliva den, for future feeding (Martin 1981; George et al. 1986; 40 Dufton 1992). peptide has the amino acid sequence shoWn in FIG. 1A or The shreW venom literature generally consists of seven derivatives thereof, such as the peptide in FIG. 1B and other articles from the 40s and 50s and one MA thesis in 1966 derivatives described herein. The invention includes meth [Christenbury 1966]. These are summarized in a revieW ods of isolating a paralytic compound from venomous shreW saliva gland or shreW saliva, comprising providing the gland [Dufton 1992]. Using a crude ammonium sulfate precipitate 45 of shreW saliva glands, Ellis and Krayer (1955) concluded or saliva, isolating the paralytic compound from the gland or the active agent Was probably a protein and, because of its saliva and optionally purifying the compound. inability to dialyZe, a larger protein. A major contribution of The shreW submaxillary gland or saliva is optionally the Ellis & Krayer Work Was to shoW activity in cats, dogs, isolated from Blarina brevicauda, Blarina carolinensis, mice, rats, guinea pigs and rabbits. Christenbury [1966] 50 Sorex unguiculalus, Sorex Sh/I’IZO saevu (Solenodon para shoWed Ellis & Krayer’s preparation stopped oxygen con doxus), Neomys fodiens, Sorex marilimensis or Neomys sumption by mouse kidney and liver slices. Japanese patent anomalous. The peptide is potent, for example, i) a 10 application (JP 10-236963; 1998) appears to disclose an microlitre dose of 20% (W/v) crude gland extract injected alcoholic extract of saliva glands from tWo shreW species into a mealWor'm in an in vitro assay causes mealWor'm (Sorex unguiculalus & Sorex Sh/I’IZO saevus) as a calcium 55 paralysis in less than 1 second; and ii) a 10 microlitre dose channel blocker and its use as a hypotensive. The purity is of 10% (W/v) crude gland extract injected into a mealWor'm loWithe extract includes any compounds that Would dis in an in vitro assay causes mealWor'm paralysis in less than solve in 70% ethanol. There is no information about the 10 seconds. responsible active molecule/s in the unknoWn mixture of The invention also includes peptides of the invention in a compounds. 60 puri?ed form. The peptides are optionally puri?ed at least 90%, 95% or 99%. The invention also includes an isolated SUMMARY OF THE INVENTION peptide comprising a fragment of 5-10, 10-15, 15-20, or 20-24 amino acids of a peptide described in this application. The paralytic compound of shreW saliva remained uni The invention also optionally includes pharmaceutical com denti?ed until noW. The inventors have isolated and puri?ed 65 position or cosmetic composition or insecticide composition a paralytic compound having the sequence shoWn in FIG. 1A including a peptide of the invention. The invention further (SEQ ID N011) and identi?ed derivatives, such as the optionally includes an isolated and puri?ed multiprotein US 7,273,850 B2 3 4 complex comprising the peptide of claims 1 or 2 and having Within the spirit and scope of the invention Will become a molecular Weight of greater than or equal to 600,000 apparent to those skilled in the art from the detailed descrip daltons. tion. Another aspect of the invention comprises a method of BRIEF DESCRIPTION OF THE DRAWINGS dissociating the peptide of the invention from a multiprotein complex described herein, comprising contacting the mul These and other features of embodiments of the invention tiprotein complex With sodium docecylsulfate or aqueous Will become more apparent from the folloWing description alcohol or Warming at 400 C. in Which reference is made to the appended draWings The present invention also provides a pharmaceutical Wherein: composition or a cosmetic composition that includes the FIG. 1. Amino acid sequences: A. (SEQ ID NOzl); B. isolated and puri?ed shreW saliva peptide, and the use of the (SEQ ID NO:2). peptide as a pharmaceutical substance, neuromuscular FIG. 2. SiZe exclusion chromatography of shreW submax blocker or an analgesic, for example, as an analgesic for ilary gland extract With bioactive fractions indicated by Wounds. The invention is yet further directed to the use of cross-hatching. the isolated and puri?ed shreW saliva peptide for prevention FIG. 3. SDS-PAGE analysis of shreW submaxilary gland or treatment of migraine, myofacial and other types of pain, extract. The small active component exists as part of a very muscle tremors, neuromuscular diseases, excessive sWeating high molecular Weight complex. and Wrinkles. The invention also optionally relates to the use FIG. 4. First HPLC elution pro?le of active fraction. of a peptide of the invention descried herein as an insect 20 FIG. 5. Second HPLC elution pro?le of active fraction. immobilizing agent or an insecticide. A peptide shoWn in FIG. 6. SDS-PAGE gel of both buccal saliva and sub FIG. 1A or 1B Would be an example of a compound for all maxilary homogenate stained for glycoproteins. the aforementioned uses. FIG. 7. SDS-PAGE gel Coomassie stain of both buccal saliva and submaxilary homogenate. In particular, the invention is directed to a method of preventing or treating migraines, myofacial and other types FIG. 8. Capillary electrophoretogram of the isolated and puri?ed shreW saliva peptide in sodium borate buffer. of pain, muscle tremors, neuromuscular diseases, and exces FIG. 9. Capillary electrophoretogram of the isolated and sive sWeating in a mammal comprising administering to the puri?ed shreW saliva peptide. mammal an isolated and puri?ed shreW saliva peptide, for FIG. 10. Ultra-violet spectrum of the isolated and puri?ed example in a pharmaceutical composition. The mammal is shreW saliva peptide. preferably a human. The invention is also directed to a 30 FIG. 11. MALDI-TOF mass spectrum of the isolated and method of providing analgesia, for example, for an analgesic puri?ed shreW saliva peptide. for Wounds, or neuromuscular blocking in a mammal com FIG. 12. Peptide mass mapping of tryptic peptides of the prising administering to a mammal a pharmaceutical com position including the isolated and puri?ed shreW saliva isolated and puri?ed shreW saliva peptide. FIG. 13. MASCOT searching results of the MS/MS data peptide. The invention is further directed to a method of 35 from HPLC-ESI-Q-TOF analysis. preventing or reducing Wrinkles in a mammal comprising FIG. 14. MealWorms immediately post-inj ection and With administering to the mammal the isolated and puri?ed shreW total paralysis. saliva peptide, for example in a cosmetic composition. The FIG. 15. Migration time vs isoelectric pH of Beckman invention is also directed to a method of killing or immo Coulter pI standard proteins. biliZing an insect comprising administering to the insect a 40 peptide of the invention, for example in an insecticidal FIG. 16. The increased ?uorescence due to calcium ion composition, for example, by infecting insects With species uptake by ovarian carcinoma cell line OV-2008 and subse speci?c viruses engineered to direct the infected insect to quent formation of the FURA/Ca+2 in the absence (control) produce the paralytic peptide (a useful virus is a Baculovi and presence of the paralytic shreW peptide soricidin after initial treatment With calcium chloride (to a ?nal concen rus). A peptide shoWn in FIG. 1A or 1B or another com pound described herein Would be an example of a compound tration of 2.5 mM), potassium chloride (to 20 mM) and a suitable for all the aforementioned methods. ?nal calcium chloride (to a ?nal concentration of 5.0 mM) treatments. The invention is also directed to the use of the isolated and FIG. 17. The increased ?uorescence due to calcium ion puri?ed shreW saliva peptide for the preparation of antibod 50 uptake by insect nerve tissue and subsequent formation of ies, including polyclonal antibodies, monoclonal antibodies the FURA/Ca+2 in the absence (control) and presence of the or functional fragments thereof. This invention also relates paralytic shreW peptide soricidin after a challenge With to the antibodies so produced. calcium chloride (to a ?nal concentration of 2.5 mM) and The invention is yet further directed to a method of potassium chloride (to 20 mM). determining the potency of a paralytic agent by administer 55 ing the paralytic agent to a mealWorm or other insect; DETAILED DESCRIPTION OF THE determining the time until onset of paralysis and/or the INVENTION duration of paralysis; and Wherein the time for onset of paralysis is inversely proportional to the strength of the The invention involves isolation and puri?cation of a paralytic agent and the duration of paralysis is proportional 60 peptide paralytic agent from shreW salivary gland or saliva to the strength of the paralytic agent. (called “PS peptide” or soricidin). The peptide preferably Other features and advantages of the present invention has 54 amino acids and the sequence shoWn in FIG. 1A Will become apparent from the folloWing detailed descrip (SEQ ID NO:l) or is a derivative, such as that shoWn in FIG. tion. It should be understood, hoWever, that the detailed 1B (SEQ ID NO:2) or another derivative as described description and the speci?c examples While indicating pre 65 herein. In one embodiment, FIG. 1A is native isolated ferred embodiments of the invention are given by Way of sequence and 1B is a derivative of native sequence. Option illustration only, since various changes and modi?cations ally the amino acid sequences are isolated and puri?ed. The US 7,273,850 B2 5 6 peptide may be isolated from any shrew having paralytic (increased ionic strength does not dissociate it) and prefer activity in its saliva, such as Blarina, Neomys and Sorex ably is exposed to treatment With sodium dodecylsulfate shreW species. The invention also optionally includes a (SDS) or With aqueous ethanol to dissociate it from the bioassay using the common mealWorm or other insect for complex. Any short chain alcohol (preferably C1 to C6, rapid assessment of paralytic bioactivity. For example, the more preferably C2 or C3) such as isopropyl alcohol, bioassay shoWs that paralytic saliva administered to the propanol or butanol may be used in place of ethanol. It mealWorm can keep it paralyZed but alive for at least 7 days. appears that the bioactive peptide is kept complexed in the The toxin is very powerful; in dose response studies a 10 salivary gland until it is released as an active form in the microlitre injection of 20% (W/v) crude gland extracts saliva. The production of active peptide can be increased, for produces total paralysis in less than 1 sec While 10% requires example, by ?rst preparing a cold acetone precipitation (eg. 10 sec for total paralysis. The 10 microlitre sample repre a pH 7 phosphate bu?fered 10% (W/v) homogenate of the sented about 8 micrograms of total soluble extracted protein submaxilary gland) or other suitable suitable precipitation (0.8 mg/mL of extract, 0.010 mL of this injected:0.008 solvent, dissolving the dried solid acetone precipitate (eg. in mg:8 micrograms total soluble protein). Of this, the peptide pH 7 phosphate bulfer) and incubating (eg. at 370 C. for 20 represents (as assessed from the gel stain density) about 1/10 minutes). This treatment increases the release of active of the protein in the Whole extract (far right lane of gel peptide from the complex containing it. As Well, the pure picture). Thus, the actual peptide injected represents about peptide can be isolated by preparative HPLC or other 0.8 micrograms of material or 800 nanograms. Using the separation methods. The peptide isolate is reactive With bioassay and various chromatographic methods the inven Clellands reagent indicating the presence of sulfhydryl tors isolated a peptide(s) With a molecular Weight of about 20 groups and the amino acid cysteine although it is reasonable 6000 (SDS-PAGE) that shoWs paralytic activity. Unexpect to expect these to exist in disulfhydryl bonds. The peptide edly, the small active component exists as part of a very high preparation also shoWed an absorbance at 280 nm indicating molecular Weight, multiprotein complex (FIG. 2; FIG. 3, the presence of aromatic amino acids. In particular, the lane 1) the molecular Weight of the complex Was about peptide preparation shoWed Weak absorption at 280 nm, but 600,000 daltons. It appeared in a void volume fraction from 25 stronger absorption at 260 nm, indicating phenylalanine but a siZe exclusion column (Sephadex G-200) that has a not tyrosine and tryptophan. FIG. 14 shoWs mealWorms molecular Weight cut-off of 600,000 daltons. After puri?ca immediately post-injection and With total paralysis. tion, the complex shoWs a single band on the gel (FIG. 3 lane The peptide may be modi?ed as described beloW to 2). The peptide sequence is readily obtained by knoWn produce variants of the paralytic peptide with different techniques, such as the standard sequential Edman degra 30 paralytic potencies. Some variants that Will be developed by dation (P. Edman and G. Begg. 1967. Eur. J. Biochem. 11 this process Will have the potential to behave as competitive 80-91. H. D. Niall, 1973. Methods EnZymol. 27: 942-1010.) inhibitors (e.g. antidotes) to paralysis developed in response and mass spectroscopic sequence determination. to our peptide. The secondary structure of soricidin, based on tWo dimen Peptides of the Invention sional structure analysis shoWs an alpha/beta sca?fold. The 35 disul?de linkage pattern (2-23; 6-27; 9-41) Was determined The invention provides an isolated PS peptide. The term by comparison With mammalian synenkephalin (Lecchi et “PS peptide” as used herein includes the peptides shoWn in al., 1997) Whose amino acid sequence is 52% homologous FIG. 1A (SEQ ID N011) or FIG. 1B (SEQ ID N012), With soricidin further supports this structure prediction by homologs, analogs, mimetics, fragments or derivatives of providing rationale for stabiliZation of the alpha/beta topol 40 the PS peptide. ogy. The peptide architecture, based on topology homology In one embodiment, the isolated PS peptide consists of 54 With the Chinese scorpion (Bulhus marlensi) toxin amino acid residues and has the sequence shoWn in FIG. 1A BmBKTxl (no sequence homology With soricidin) (Cai et (SEQ ID N011) or the derivative shoWn in FIG. 1B (SEQ ID al., 2004), shoWs a calcium-activated potassium ion channel N012). In another embodiment, the PS peptide comprises blocker and a mechanism of paralysis similar to that dis 45 sequences substantially identical to the above-noted pep played by this family of non-homologous scorpion neuro tides or comprising an obvious chemical equivalent thereof. toxins. It also includes peptide sequence plus or minus amino acids Thus the invention includes a method of isolating and at the amino and/ or carboxy terminus of the above-noted PS sequencing a paralytic shreW peptide by isolating the peptide peptide sequences. In yet another embodiment, the invention as described in this application and sequencing the peptide. 50 includes fusion proteins, comprising the PS peptide, labeled In one embodiment, the sequence of the isolated and puri?ed PS peptides, analogs, homologs and variants thereof. shreW saliva peptide is shoWn in FIG. 1A (SEQ ID N011) Within the context of the present invention, a peptide of and a derivative sequence is shoWn in FIG. 1B (SEQ ID the invention may include various structural forms of the N012) and the invention includes other derivatives of the primary PS peptide Which retain biological activity. For sequence as described herein. TWo examples of methods that 55 example, a peptide of the invention may be in the form of are optionally used to isolate the protein are: i) siZe exclu acidic or basic salts or in neutral form. In addition, indi sion and ion exchange chromatography and ii) centrifuga vidual amino acid residues may be modi?ed by oxidation or tion through membranes With distinct molecular Weight reduction. cut-olfs: preferably 100,000, 10,000 and 3,000 Dalton In addition to the full-length amino acid sequence, the molecular Weight cut-olf Centricons from Amicon. Other peptide of the present invention may also include trunca methods are also useful. The ?rst method alloWs separation tions, analogs and homologs of the peptide and truncations of the complexed active agent (very high molecular frac thereof as described herein. Truncated peptides or fragments tions) from Where a free peptide of molecular Weight 6000 may comprise peptides of at least 5, 10, 15, 20, 25, 30, 35, Would normally elute from the siZe exclusion chromatogra 40, 45 or 50 amino acids or more amino acid residues of the phy column. The ion exchange chromatographic protocols 65 sequence listed above. Useful fragments also include, for employed a anion exchanger of a sodium phosphate bulfer, example, 50-54, 45-50, 45-52, 44-55, 42-54, 40-54, 35-45 or neutral pH. The peptide is strongly bound to the complex 25-35 amino acids. Useful fragments are capable of provid