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(12) United States Patent (10) Patent No.: US 9,174.999 B2 Du Bois Et Al

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(12) United States Patent (10) Patent No.: US 9,174.999 B2 Du Bois Et Al US009 174999B2 (12) United States Patent (10) Patent No.: US 9,174.999 B2 Du Bois et al. (45) Date of Patent: Nov. 3, 2015 (54) METHODS AND COMPOSITIONS FOR Arakawa O, Nishio S. Noguchi T. Shida Y and Onoue Y. A New STUDYING, IMAGING, AND TREATING PAIN Saxitoxin Analogue from aXanthid Crab Atergatis Floridus. Toxicon 1995; 33:1577-1584. (75) Inventors: Justin Du Bois, Palo Alto, CA (US); Dell'Aversano C. Walter JA, Burton IW. Stirling DJ, Fattorusso E and John Mulcahy, Stanford, CA (US); Quilliam MA. Isolation and Structure Elucidation of New and Brian Andresen, Menlo Park, CA (US); Unusual Saxitoxin Analogues from Mussels. J. Nat. Prod. 2008; David C. Yeomans, Sunnyvale, CA T1:1518-1523. (US); Sandip Biswal, Stanford, CA (US) Vale P. Metabolites of saxitoxin analogues in bivalves contaminated by Gymnodinium catenatum. Toxicon 2010; 55:162-165. (73) Assignee: The Board of Trustees of the Leland Koehn FE. Hall S, Wichmann CF, Schnoes HK, Reichardt PB. Stanford Junior University, Palo Alto, Dinoflagellate neurotoxins related to saxitoxin: structure and latent CA (US) activity of toxins B1 and B2. 1982; 23:2247-2248. Onodera H, Satake M, Oshima Y. Yasumoto T and Carmichael W.W. (*) Notice: Subject to any disclaimer, the term of this New Saxitoxin Analogues from the Freshwater Filamentous patent is extended or adjusted under 35 Cyanobacterium Lyngbya wollei. Natural Toxins 1997; 5:146-151. U.S.C. 154(b) by 277 days. Vale P. New Saxitoxin analogues in the marine environment: devel opments in toxin chemistry, detection and biotransformation during (21) Appl. No.: 12/800,053 the 2000s. Phytochem Rev 2010; 9:525-535. Yotsu-Yamashita M. Kim YH. Dudley SC, Choudhary G, Pfahnl A. (22) Filed: May 7, 2010 Oshima Y. Daly JW. The structure of Zetekitoxin AB, a saxitoxin analog from the Panamian golden frog Atelopus Zeteki: a potent (65) Prior Publication Data sodium-channel blocker. PNAS 2004; 101:4346-4351. Hall S, Darling SD, Boyer GL, Reichardt PB, Liu HW. Dinoflagellate US 2010/02849 13 A1 Nov. 11, 2010 neurotoxins related to saxitoxin: structures of toxins C3 and C4, and confirmation of the structure of neosaxitoxin. Tet Lett 1984; 25:3537 3538. Related U.S. Application Data (Continued) (60) Provisional application No. 61/176,172, filed on May 7, 2009. Primary Examiner — Rebecca Anderson (51) Int. Cl. Assistant Examiner — Karen Cheng CO7D 487/4 (2006.01) (74) Attorney, Agent, or Firm — David A. Roise; VLP Law (52) U.S. Cl. Group LLP CPC .................................... C07D487/14 (2013.01) (58) Field of Classification Search CPC ............................ A61K31/519; C07D 487/16 (57) ABSTRACT USPC ........................................... 544/251514/267 See application file for complete search history. Saxitoxin analog compounds, compositions, pharmaceutical compositions, methods of synthesis of saxitoxin analogs, (56) References Cited methods of imaging, methods of treatment, including meth ods of treating pain, are provided. Saxitoxin (STX), gonyau U.S. PATENT DOCUMENTS toxin (GTX), and Zetekitoxin, and variant STX compounds bind to sodium channels and are effective to reduce or block 3,957,996 A 5, 1976 Adams et al. 6,030,974 A 2/2000 Schwartz et al. flow of Sodium ions through Such channels. Such channel 6,326,020 B1 12/2001 Kohane blockaffects nerve and muscle action, and may be effective to 2002fO161013 A1 10, 2002 Liu et al. reduce or block pain sensations, relax muscles, reduce muscle 2005/O137177 A1 6, 2005 Shafer spasm, and reduce wrinkles. STX analog binding to Sodium 2005/0202093 A1 9, 2005 Kohane et al. 2006/0057647 A1* 3/2006 Robillot ......................... 435/7.5 channels may also be useful to locate, image, or mark Sodium 2007/028O970 A1 12, 2007 Wilson channels, and so be useful in studying Sodium channels and 2008, 0021051 A1 1/2008 Wilson Sodium channel disorders, and in the diagnosis and treatment 2008.OO45553 A1 2/2008 Wilson of patients suffering from Sodium channel disorders. In embodiments, the variant STX compounds include conju FOREIGN PATENT DOCUMENTS gates having increased serum half-life as compared to STX when administered to a subject. EP O857.972 * 8/1998 WO O3OO6507 A1 1, 2003 In embodiments, the present disclosure provides a method for WO 2010/027641 A2 3, 2010 alleviating pain in a subject in need of treatment, the method comprising administering to the Subject an effective amount OTHER PUBLICATIONS of a saxitoxin analog compound, or a pharmaceutically Biotinylation, http://www.piercenet.com/browse. acceptable salt, isomer, tautomer or prodrug thereof, whereby pain in said Subject is alleviated. retrieved Apr. 5, 2012.* Bundgaard, Design of Prodrugs, 1985, Elsevier, Chapter 1, p. 1-4.* Han, Targeted Prodrug Design to Optimize Drug Deliver, 2000, 10 Claims, 12 Drawing Sheets AAPS Pharmsci, vol. 2(1), p. 1-11.* (4 of 12 Drawing Sheet(s) Filed in Color) US 9,174.999 B2 Page 2 (56) References Cited Jacobi PA, MartinelliMJ, Polanc S. Total Synthesis of +/-Saxitoxin. JAm Chem Soc 1984; 106:5594-5598. OTHER PUBLICATIONS International Search Report and Written Opinion dated Jan. 25, 2011. Negri, et al., “Three Novel Hydroxybenzoate Saxitoxin Analogues Negri A. Stirling D, Quilliam M. Blackburn S. Bolch C. Burton I. Isolated From the Dinoflagellate Gymnodinium Catenatum.” Chemi Eaglesham G. Thomas K. Walter J and Willis R. Three novel cal Research in Toxicology, 2003, vol. 16, pp. 1029-1033. hydroxybenzoate saxitoxin analogues isolated from the dinoflagel Onodera, et al., “New Saxitoxin Analogues from the Freshwater Filamentous Cyanobacterium Lyngbya wollei.” Natural Toxins, late gymnodinium catenatum. Chem Res Toxicol 2003; 16:1029 1997, vol. 5, pp. 146-151. 1033. Dell Aversano, et al., “Isolation and Structure Elucidation of New and Shimizu Y. Kobayashi M. Genenah A and Oshima Y. Isolation of Unusual Saxitoxin Analogues from Mussels.” Journal of Natural side-chain sulfated saxitoxin analogs. Tetrahedron 1984; 40:539 Products, 2008, vol. 71, pp. 1518-1523. 544. Shimuzu, et al., “Toxigenesis and Biosynthesis of Saxitoxin Ana Harada T. Oshima Y. Ysumoto T. Natural occurrence of logues.” Pure and Applied Chemistry, 1986, vol. 58, No. 2, pp. decarbamoylsaxitoxin in tropical dinoflagellate and bivalves. Agric 257-262. Biol Chem 1983: 47:191-193. Arakawa et al. (1994) “Occurrence of carbamoyl-N-hydroxy deriva Llewellyn LE. Saxitoxin, a toxic marine natural product that targets tives of saxitoxin and neosaxitoxin in a xanthid crab Zosimus aeneus' a multitude of receptors. Nat Prod Rep 2006; 23:200-222. Toxicon 32:175-183. Shimizu Y, Hsu C, Genenah A. Structure of saxitoxin in solutions and Schlager et al. (1996) “Micromole scale biotinylation of saxitoxin stereochemistry of dihydrosaxitoxins. J Am Chem Soc 1981; (STX) for use as a screening moiety for peptide binding libraries' 103:605-609. 1996 Medical Defense Bioscience proceedings May 12-16, 3:1590 Robillot C. Kineavy D, Burnell J. Llewellyn LE. Synthesis of bifunc 1597. tional saxitoxin analogues by biotinylation. Toxicon 2009; 53:460 Zaman et al. (1998) "Occurrence of a methyl derivative of saxitoxin 465. in Bangladeshi freshwater puffers' Toxicon 36:627-630. Strichartz GR, Hall S. Magnani B, Hong CY. Kishi Y and Debin JA. Sato, et al., “Identification of thioether intermediates in the reductive The potencies of synthetic analogues of Saxitoxin and the absolute transformation of gonyautoxins into saxitoxins by thiols'. stereoselectivity of decarbamoyl saxitoxin. Toxicon 1995; 33:723 Bioorganic & Medicinal Chemistry Letters, vol. 10, No. 6, Aug. 21. 737. 2000, pp. 1787-1789. Myasoedov NF. ShevchenkoVP. Nagaev IY, Susan A.Tritium-traced European Supplemental Search Report mailed on Jan. 16, 2013 in saxitoxin dihydrochloride and method for the production thereof. European Patent Application No. 10772881.8. U.S. Pat. No. 7,576,202. Walls, et al., “Synthesis and biological evaluation of a fluorescent Mao H. Fieber L.A. Gawley RE. Novel modulator of NaV1.1 and analog of phenytoin as a potential inhibitor of neuropathic pain and NaV1.2 Na+ channels in rat neuronal cells. Med Chem Lett 2010; imaging agent'. Bioorg. Med. Chem. Jul. 3, 2012, vol. 20, pp. 1:135-138. 5269-52.76. Iwamoto O, Shinohara R, Nagasawa K. Total synthesis of (-)- and Anderson, et al., “Voltage-gated Sodium channel blockers as (+)-decarbamoyloxysaxitoxin and (+)-Saxitoxin. Chem Asian J cytostatic inhibitors of the androgen-independent prostate cancer cell 2009; 4:277-285. line PC-3”, Mol. Canceer Ther. Nov. 14, 2003, vol.2, pp. 1149-1154. Tanino H. Nakata T. Kaneko T. Kishi Y. A Stereospecific Total Syn thesis of d.l-Saxitoxin. JAm Chem Soc 1977;99; 2818-2819. * cited by examiner U.S. Patent Nov. 3, 2015 Sheet 2 of 12 US 9,174.999 B2 ge wereapplied a sa e o bepotential eso be a owie o-week 10. Figure 3A Figure 3B Figure 3. Current recordings at varying concentrations of inhibitor on rNav1.4 expressed in CHO cells. Dose-response curves for (+)-STX 1 (top) and 2 (bottom). Figure 3 NMs NMbs H.N. NH 1. TrocC HN NH, O / soH 2. Prnet he hg -N- www.se N amwe 'NO MeO i.e." N -snubs 2.25°'sOxce N-N-kNMbs O 3 4. H2N+ Mt. RHN, -NH on HR Ho,h NH2N-- RNHTHF O'') 1. B(O2CCF) s'Yo1 NHR 23 °C N. NH was was N. NH y 2. DMSO, DCC r +NH NMts S: R is - s 7: R' se O Figure 4 U.S. Patent Nov. 3, 2015 Sheet 3 of 12 US 9,174.999 B2 O R Cso (nM) HO 'NH2 o' NHR Cah 13 8 26 at 3 -N-N-NH iPr 9. 33 at 13 HOHNN Cs-12NH 10 19 - O.8 - N -- O CshioCO2 11 135 - 7 2 rifico 12 87 - 9 O Recorded ICso values for STX derivatives against rNav1.4. Figure 5 O O * N- o'n o'n 2SctO p-FCshCO-NHS - O HN,NH CHCN/H2O ill H2N + NH * pH = 9.5 O f 13: Cso a 46+ 7 nM A final-step ligation strategy for STX modification Figure 6 U.S.
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