US009220785B2 (12) United States Patent (10) Patent No.: US 9.220,785 B2 Adami et al. (45) Date of Patent: Dec. 29, 2015 (54) LIPOPEPTIDES FOR DELIVERY OF 47/48323 (2013.01); A61 K48/0033 (2013.01); NUCLECACDS A61K 48/004I (2013.01); C07K 14/00 (2013.01); C12N 15/87 (2013.01); A61 K48/00 (71) Applicant: Marina Biotech, Inc., Bothell, WA (US) (2013.01) (72) Inventors: Roger C. Adami. Bothell, WA (US); (58)58) tField OSSOof Classification SearchSea Militigrists See application file for complete search history. WA (US) (56) References Cited (73) Assignee: Marina Biotech, Inc., Bothell, WA (US) U.S. PATENT DOCUMENTS (*) Notice: Subject to any distic the t 6,713,078 B2 * 3/2004 Lehrer et al. .................. 424/405 patent 1s extended or adjusted under 2006/0272049 A1* 11/2006 Waterhouse et al. ......... 800,279 U.S.C. 154(b) by 0 days. 2007/01293.05 A1 6/2007 Divita et al. .................... 514f13 (21) Appl. No.: 13/715,768 FOREIGN PATENT DOCUMENTS (22) Filed: Dec. 14, 2012 WO WOO198362 A2 * 12/2001 WO WO 200709965.0 A1 * 9, 2007 (65) Prior Publication Data WO WO 2008049078 A1 * 4, 2008 US 2013/0231382 A1 Sep. 5, 2013 * cited by examiner Related U.S. Application Data Primary Examiner — Jeffrey E. Russel (63) Continuation of application No. 12/750,622, filed on (74) Attorney, Agent, or Firm — Eckman Basu LLP Mar. 30, 2010, now abandoned, which is a continuation of application No. PCT/US2008/078627, (57) ABSTRACT filed on Oct. 2, 2008. (60) Provisional application No. 60/976,894, filed on Oct Lipopeptide compounds with a central peptide and having 2, 2007 pp sy--- is lipophilic groups attached at each terminus, and salts and uses s thereof. The lipophilic groups can be derived from a natu (51) Int. Cl rally-occurring lipid, or can be a C(1-22)alkyl, C(6-12)cy A6 IK 47/42 (2006.01) cloalkyl, C(6-12)cycloalkyl-alkyl, C(3-18)alkenyl, C(3-18) C07K I4/00 (2006.01) alkynyl, C(1-5)alkoxy-C(1-5)alkyl, or a sphinganine, or (2R, A6 IK 4.8/00 (2006.015 3R)-2-amino-1,3-octadecanediol, icosasphinganine, A6 IK 47/48 (2006.015 sphingosine, phytosphingosine, cis-4-sphingenine. The com CI2N 15/87 (200 6. 01) pounds can be used in compositions to deliver active agents to (52) U.S. Cl cells, including interfering-RNA agents and antisense RNAS. CPC ........... A61K 47/42 (2013.01); A61K 47/48046 (2013.01); A61 K47/48053 (2013.01); A61 K 11 Claims, No Drawings US 9.220,785 B2 1. 2 LIPOPEPTIDES FOR DELVERY OF Moreover, there is a need for compositions and methods for NUCLECACDS delivery of interfering RNAs to selected cells, tissues, or compartments to modulate gene expression in a manner that SEQUENCE LISTING will alter a phenotype or disease state. This application includes a Sequence Listing Submitted BRIEF SUMMARY herewith via EFS-Web as an ASCII file created on Apr. 2, 2013, named MDR-07-23USB1 SeqList corrected.TXT, This invention satisfies these needs and fulfills additional which is 130,036 bytes in size, and is hereby incorporated by objects and advantages by providing a range of novel com reference in its entirety. 10 pounds, compositions, formulations and methods that employ an interfering nucleic acid or precursor thereof in FIELD OF THE INVENTION combination with various components including lipopep tides, lipids, and natural or synthetic polymers. This invention relates to novel drug delivery enhancing In some embodiments, this invention includes a peptide agents including lipids that are useful for delivering various 15 having 2 to 100 amino acid residues and a lipophilic group molecules to cells. This invention provides a range of com attached to at least one terminus of the peptide, or to at least pounds, compositions, formulations, methods and uses of one amino acid residue, and salts and uses thereof. The lipo Such agents directed ultimately toward drug delivery, thera philic group may be attached to the N-terminus, C-terminus peutics, and the diagnosis and treatment of diseases and con or both termini of the peptide. The lipophilic group may be ditions, including those that respond to modulation of gene attached to at least one interal amino acid residue (i.e., an expression or activity in a subject. More specifically, this amino acid residue that is not the N-terminus or the C-termi invention relates to compounds, liposomes, lamellar vesicles, nus amino acid residue of the peptide). The lipophilic group emulsions, micelles, Suspensions, particles, solutions and may be attached to either termini or both and at least one other forms of delivery enhancing compositions and formu internal amino acid residue. lations, as well as therapeutic methods and uses for these 25 This Summary, taken along with the detailed description of delivery materials. the invention, as well as the appended examples and claims as a whole encompasses the disclosure. BACKGROUND DETAILED DESCRIPTION The delivery of a therapeutic compound to a subject can be 30 impeded by limited ability of the compound to reach a target This invention relates generally to the fields of RNA inter cell or tissue, or by restricted entry or trafficking of the com ference, delivery of RNA therapeutics, and to the chemistry of pound within cells. Delivery of a therapeutic material is in lipids. More particularly, this invention relates to composi general restricted by membranes of cells. These barriers and tions and formulations for ribonucleic acids, and their uses for restrictions to delivery can result in the need to use much 35 medicaments and for delivery as therapeutics. This invention higher concentrations of a compound than is desirable to relates generally to methods of using ribonucleic acids in achieve a result, which brings the risk of toxic effects and side RNA interference for gene-specific inhibition of gene expres effects. sion in mammals. One strategy for delivery is to improve transport of a com This invention provides a range of compositions, formula pound into cells using lipid or polymeric carrier molecules. 40 tions and methods which include an interfering nucleic acid These materials can take advantage of mechanisms that exist or a precursor thereof in combination with various compo for selective entry into a cell, while still excluding exogenous nents including lipopeptides, lipids, lipoid moieties, and molecules such as nucleic acids and proteins. For example, a natural or synthetic polymers. cationic lipid may interact with a drug agent and provide In some aspects, this invention provides novel composi contact with a cell membrane. Lipid molecules can also be 45 tions to facilitate the delivery of nucleic acids including organized into liposomes or particles as carriers for drug RNAi-inducing agents, antisense RNA agents, or DNA to agents. Liposomal drug carriers can protect a drug molecule cells, tissues, organs, and in living animals, for example, from degradation while improving its uptake by cells. Also, mammals and humans. liposomal drug carriers can encapsulate or bind certain com Lipopeptides pounds by electrostatic and other interactions, and may inter 50 This invention provides a range of lipopeptides for delivery act with negatively charged cell membranes to initiate trans and administration of interfering-RNA agents or antisense port across a membrane. RNAS. The lipopeptides can be cationic, or non-cationic. As The understanding of regulatory RNA and the develop used herein, non-cationic includes neutral and anionic. Cat ment of RNA interference (RNAi), RNAi therapy, RNA ionic lipopeptides may have one or more cationic sites. based drugs, antisense therapy, and gene therapy, among oth 55 Lipopeptides of this disclosure may be formed by substi ers, has increased the need for effective means of introducing tuting an nucleic acid delivery-enhancing or lipoid group at active nucleic acid the N-terminus or the C-terminus of a peptide, or both termini agents into cells. In general, nucleic acids are stable for of the peptide. In some aspects, the nucleic acid delivery or only limited times in cells or plasma. However, nucleic acid lipoid group may be linked to the alpha-carbon of one or more based agents can be stabilized in compositions and formula 60 an amino acid residues of the peptide or to one or more amine tions which may then be dispersed for cellular delivery. groups, each amine group being adjacent to the alpha-carbon What is needed are compositions and formulations for of an amino acid residue of the peptide, or a combination of intracellular and in vivo delivery of a nucleic acid agent for the N-terminus, C-terminus, alpha-carbon of one or more an use, ultimately, as a therapeutic, which maintain cytoprotec amino acid residues of the peptide or to one or more amine tion and relatively low toxicity. Furthermore, there is a need 65 groups, each amine group being adjacent to the alpha-carbon for compositions and methods to deliver double-stranded of an amino acid residue of the peptide. In some embodi RNA to cells to produce the response of RNA interference. ments, the peptide core may include two or more sequential US 9.220,785 B2 3 4 amino acids, or a peptide of 2 to 100 amino acid residues (or Lipopeptides of this disclosure can be prepared where, for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, example, the central peptide may be Poly-Lys-Trp., 4:1, M. 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34,35, 36, 37, 20,000-50,000; Poly-Orn-Trp., 4:1, M20,000-50,000; frag 38,39, 40, 41,42, 43,44, 45,46, 47, 48,49, 50, 51, 52,53,54, ments or variants of mellitin protein, and fragments or vari 55,56, 57,58, 59, 60, 61, 62,63, 64, 65, 66, 67,68, 69,70, 71, ants of a histone protein, e.g., histone H1, histone H2A, 72, 73,74, 75,76, 77,78, 79,80, 81,82, 83, 84,85, 86, 87,88, histone H2B, histone H3 or histone H4.