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WO1997046671A1 - Efficacite amelioree d'apport liposomal de molecules anti-sens - Google Patents

Efficacite amelioree d'apport liposomal de molecules anti-sens Download PDF

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WO1997046671A1
WO1997046671A1 PCT/CA1997/000347 CA9700347W WO9746671A1 WO 1997046671 A1 WO1997046671 A1 WO 1997046671A1 CA 9700347 W CA9700347 W CA 9700347W WO 9746671 A1 WO9746671 A1 WO 9746671A1
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Sandra K. Klimuk
Sean C. Semple
Peter Scherrer
Michael J. Hope
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University Of British Columbia
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Priority to CA002256456A priority Critical patent/CA2256456A1/fr
Priority to JP10500030A priority patent/JP2000511541A/ja
Priority to EP97921565A priority patent/EP0906421A1/fr
Publication of WO1997046671A1 publication Critical patent/WO1997046671A1/fr

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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
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    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A—HUMAN NECESSITIES
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Definitions

  • liposomes When made as relatively small particles (approximately 100 nm in diameter), liposomes will passively accumulate at sites of inflammation by moving through the restructured vasculature. Numerous studies have determined that liposomes or lipid complexes have the ability to deliver oligonucleotides intracellularly through two mechanisms: cellular uptake of liposomes via endocytosis, and fusion of cationic liposomes with target cell membranes.
  • Comparisons of free and encapsulated oligonucleotides indicate an enhanced stability for encapsulated oligos in vitro as the liposome prevents nuclease degradation.
  • comparison of free and encapsulated phosphorothioate oligonucleotides usually indicate no enhancement as the phosphorothioate oligos are themselves nuclease resistant.
  • the same amount of oligonucleotide can potentially be delivered to the cell whether it is a free phosphorothioate or an encapsulated phosphodiester oligonucleotide.
  • SUBSTTTUTE SHEET (RULE 26)
  • cationic lipid vesicles form "complexes" with DNA, including plasmids and oligonucleotides. These complexes are not liposomes (i.e. an intact bilayer encapsulating an aqueous space) but are aggregates of lipid and DNA held together by electrostatic attraction between the cationic lipid and anionic nucleic acid.
  • a fusogenic factor such as phosphatidylethanolamine or a fusion protein is required to achieve a significant antisense effect or gene transfection.
  • the complement system is a multi-protein cascade which serves as one of the first lines of defense against foreign particles which have entered the blood.
  • the two basic mechanisms by which complement attacks foreign particles are by opsonization and cell lysis.
  • Opsonization involves the covalent attachment of complement fragments, principally C3b and iC3b, to the surface of a particle or cell, which is then recognized by corresponding receptors present on macrophages.
  • Cell lysis involves the assembly of a multiprotein complex, C5b-9, which perforates cell membranes and generates a pore.
  • the free oligonucleotide does not appear to penetrate endothelial cells in culture but when complexed with cationic lipid/phosphatidylethanolamine (PE) complexes extensive uptake is observed.
  • PE cationic lipid/phosphatidylethanolamine
  • Such cationic lipid/PE complexes are known to disrupt intracellular endosomes and deliver nucleic acids into the cytoplasm.
  • antisense oligonucleotides rapidly diffuse into the nucleus (Sixou, et al. , Nuc. Acids Res. 22:662-668 (1994)) and this is also observed for anti ICAM-1 delivered by aggregates or complexes of the oligonucleotides and lipids (see Bennett, et al, Mol.
  • the phosphorothioates noted above, in which an oxygen atom is replaced by a sulphur in the phosphate backbone, exhibit increased resistance to nucleases and are more stable in vivo than normal phosphodiester oligonucleotides (Juliano, et al. , Antisense Research & Development 2:165-176 (1992)).
  • nucleic acid methylphosphonates which are not only nuclease resistant but also hydrophobic analogues of phosphodiesters and therefore expected to be more membrane permeable (see, Hughes, et al. , J. Pharm. Sci.
  • Attractive targets for antisense therapy include the nucleic acids which encode intercellular adhesion molecule- 1 (ICAM-1), vascular cell adhesion molecule- 1
  • ICAM-1 SUBST ⁇ UTE SHEET (RULE 26) (VCAM-1), and endothelial leukocyte adhesion molecule-1 (ELAM-1).
  • ICAM-1 which is a 90- 110 kDa membrane glycoprotein involved in the trafficking of leukocytes out of the vasculature and in antigen presentation to T cells (see Osborn, Cell 56:907-910 (1990) and Springer. Nature (Lond.) 346:425-443 (1990)). ICAM-1 is normally expressed at low levels on the surface of endothelial cells, keratinocytes, fibroblasts and leukocytes.
  • ICAM-1 is inducible by a number of cytokines, including IL- lj3, tumor necrosis factor- ⁇ and interferon- ⁇ . Increased expression of ICAM-1 has been demonstrated in a variety of human diseases and has been shown to correlate with leukocyte infiltration in the diseased tissue. What is needed in the an are new compositions and methods for the delivery of antisense molecules directed toward inhibiting the expression of cellular adhesion molecules. Such compositions should increase the serum stability of the antisense molecules and reduce toxic side effects such as complement activation. Surprisingly, the present invention provides such compositions and methods.
  • the present invention provides pharmaceutical compositions for the treatment of pathologic conditions associated with the overexpression of cellular adhesion molecules, such as ICAM-1 in a host.
  • These pharmaceutical composition comprise an effective amount of an ICAM-1 antisense molecule encapsulated in a lipid mixture which is typically a liposome or lipid particle.
  • the lipid mixture will typically comprise at least two members selected from the group consisting of phosphoiipids, sterols and cationic lipids.
  • the antisense molecule is either a phosphorothioate molecule or a methyl phosphonate molecule, from about 15 to 50 nucleic acids, and is complementary to a portion of the 3 '-untranslated region of
  • the liposome will preferably comprise phosphatidylcholine and cholesterol, more preferably egg phosphatidylcholine and cholesterol.
  • the particles will preferably comprise phosphoiipids and cationic lipids.
  • the present invention provides methods for the treatment of pathologic conditions associated with the overexpression of ICAM-1 in a host.
  • pathologic conditions include Alzheimer's disease, multiple sclerosis, uveitis, Herpes keratitis, renal allograft rejection, glomerulonephritis, liver allograft rejection, viral hepatitis, alcoholic hepatitis, cholangitis, cardiac allograft rejection, atherosclerotic plaques, rheumatoid arthritis, Grave's disease, Hashimoto's thyroiditis, psoriasis, scleroderma, graft v host disease, contact dermatitis, lichen planus, fixed drug eruption, mycosis fungoides, and alopecia areata.
  • Figure 1 illustrates a spin column elution profile of encapsulated antisense. 50 ⁇ L of encapsulated antisense was applied to a 1 mL Biogel A15m, 200-400 mesh, spin column and separated. Lipid was determined by phosphate analysis (O), and oligonucleotide was detected by measuring A 260 , after Bligh and Dyer extraction (•).
  • O phosphate analysis
  • oligonucleotide was detected by measuring A 260 , after Bligh and Dyer extraction (•).
  • Figure 2 illustrates a purified liposomal antisense preparation.
  • Liposome- encapsulated antisense was "purified” on DEAE-sepharose CL-6B columns. Removal of free antisense was assessed by size exclusion chromatography on 1 mL Biogel A15m column. Lipid was determined by phosphate analysis (O), and oligonucleotide was detected by measuring A 260 , after Bligh and Dyer extraction (•).
  • Figure 3 shows a time course for leakage of encapsulated antisense. Leakage of encapsulated antisense was monitored at room temperature for 1 (O), 3 ( ⁇ ) and 5 (•) days.
  • Figure 4 shows the time course for leakage of encapsulated antisense at 4°C. Leakage of encapsulated antisense was monitored for 1 (O), 3 (D), and 5 (•) days, at 4 q C.
  • Figure 5 illustrates complement activation by liposomes. Complement activation was investigated using an EPC/CH liposome preparation. Liposomes were 100 ⁇ 30 nm. Lipid composition is expressed in molar ratios.
  • Figure 6 illustrates the ear swelling characteristics of ICR mice. Measurements refer to increases in ear thickness with respect to baseline measurements (prior to ear challenge). Values are given for two separate experiments. Error bars represent the standard deviation of measurements from 4 mice.
  • Figure 7 illustrates the ear swelling characteristics of BALB/c mice.
  • Measurements refer to increases in ear thickness with respect to baseline measurements (prior to ear challenge). Error bars represent the standard deviation of measurements from 4 mice.
  • Figure 8 shows the liposome accumulation in the ears of ICR mice during various time intervals of inflammation. Liposomes were injected into mice at 0 hr, 24 hr, and 48 hr after initiation of ear inflammation. Liposomes were allowed to circulate for 24 hr at which time ears were recovered, digested, and analyzed for radiolabeled lipid. Error bars represent the standard deviation of measurements from 4 mice.
  • Figure 9 shows the liposome accumulation in the ears of BALB/c mice during various time intervals of inflammation. Liposomes were injected into mice at 0 hr, 24 hr, and 48 hr after initiation of ear inflammation. Liposomes were allowed to circulate for 24 hr at which time ears were recovered, digested, and analyzed for radiolabeled lipid. Error bars represent the standard deviation of measurements from 4 mice.
  • Figure 10 shows the MPO levels in the ears of ICR mice during inflammation. At various times after the initiation of inflammation, inflamed ears ( ⁇ ) and control ears (•) were recovered, homogenized, and assayed for MPO activity. Error bars represent the standard deviation of measurements from 4 mice.
  • Figure 11 shows cell infiltration into the inflamed ear of ICR mice during inflammation. Bone marrow cells and circulating leukocytes were labeled 24 hr prior to the onset of inflammation. At various times after the initiation of inflammation, ears were recovered, digested, and analyzed for radiolabeled cells by liquid scintillation counting. Error bars represent the standard deviation of measurements from 4 mice.
  • Figure 12 shows a typical inflammation experiment involving ICR mice. The following parameters were measured: ear swelling ( ⁇ ); liposome accumulation in inflamed (•) and non- inflamed ( ⁇ ) ears; and cell infiltration (O).
  • Figure 13 shows liposome accumulation in the ears of ICR mice during the first 24 hours of inflammation.
  • DSPC:CH liposomes were injected into mice
  • SUBSTrrUTE SHEET (RULE 26) immediately after initiation of ear inflammation. At various times mice were sacrificed and the ears were collected and analyzed (inflamed (•) and non-inflamed ( ⁇ ) ears). Error bars represent the standard deviation of measurements from 4 mice.
  • Figure 14 illustrates the circulation clearance rates of free [ 3 H]-antisense and liposome encapsulated pHJ-antisense.
  • Figure 15 illustrates the tissue biodistribution of free [ 3 H] -antisense (Isis 2302).
  • Figure 16 illustrates the tissue biodistribution profiles for both the lipid and antisense portions of a lipsome encapsulated antisense formulation.
  • Figure 17 illustrates the ability of free antisense to inhibit ear inflammation.
  • Figure 18 illustrates the efficacy of free and encapsulated ICAM-1 antisense formulations in reducing ear inflammation in mice.
  • Figure 19 is a bar graph showing edema formation (based on ear weights) in mice treated with free and encapsulated antisense.
  • PEG-Cer-C 20 l-O-(2'-( ⁇ -methoxypolyethyleneglycol)succinoyl)-2-N- arachidoyl-sphingosine
  • PEG-Cer-C 14 l-O-(2'-( ⁇ -methoxypolyethyleneglycol)succinoyl)- 2-N-myristoyl-sphingosine
  • PBS phosphate-buffered saline
  • EGTA ethylenebis(oxyethylenenitrilo)-tetraacetic acid
  • OGP n-octyl ⁇ -D-glycopyranoside (Sigma Chemical Co. , St. Louis, MO)
  • POPC palmitoyl oleoyl phosphatidylcholine
  • oligonucleotide refers to a single or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end. It includes both self-replicating plasmids, infectious polymers of DNA or RNA and non ⁇ functional DNA or RNA.
  • phosphorothioate and methyl phosphonate refer to those oligonucleotides in which a phosphodiester intemucleotide linkage has been modified by replacing at least one of the non-bridged oxygens of the intemucleotide linkage with sulfur or a methyl group, respectively.
  • Selectivity of hybridization exists when hybridization (or base pairing) occurs that is more selective than total lack of specificity.
  • selective hybridization will occur when there is at least about 55 % paired bases over a stretch of at least 14-25 nucleotides, preferably at least about 65%, more preferably at least about 75 % , and most preferably at least about 90% . See, M. Kanehisa Nucleic Acids Res. 12:203 (1984), incorporated herein by reference.
  • lipid refers to any fatty acid derivative which is capable of forming a bilayer such that a hydrophobic portion of the lipid material orients toward the bilayer while a hydrophilic portion orients toward the aqueous phase.
  • Hydrophilic characteristics derive from the presence of phosphato, carboxylic, sulfato. amino, sulfhydryl, nitro, and other like groups. Hydrophobicity could be conferred by the inclusion of groups that include, but are not limited to, long chain saturated and unsaturated aliphatic hydrocarbon groups and such groups substituted by one or more aromatic, cycloaliphatic or heterocyclic group(s).
  • Preferred lipids are phosphoglycerides and sphingolipids, representative examples of which include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoyl phosphatidylcholine, lysophosphatidylcholine, lysophosphatidyl- ethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoyl- phosphatidylcholine or dilinoleoylphosphatidylcholine could be used.
  • lipid Other compounds lacking in phosphorus, such as sphingolipid and glycosphingolipid families are also within the group designated as lipid. Additionally, the amphipathic lipids described above may be mixed with other lipids including triglycerides and sterols.
  • cationic lipid refers to any of a number of lipid species which carry a net positive charge at physiological pH. Such lipids include, but are not limited to, DODAC, DOTMA, DDAB, DOTAP, DC-Choi and DMRIE. Additionally, a number of commercial preparations of cationic lipids are available which can be used in the present invention.
  • LIPOFECTIN ® commercially available cationic liposomes comprising DOTMA and DOPE, from GIBCO/BRL, Grand Island, New York, USA
  • LIPOFECTAMINE ® commercially available cationic liposomes comprising DOSPA and DOPE, from GIBCO/BRL
  • TRANSFECTAM ® commercially available cationic liposomes comprising DOGS from Promega Corp.
  • pathologic conditions associated with the overexpression of ICAM-1 is meant to include diseases of the central nervous system (e.g. Alzheimer's disease and multiple sclerosis), the eye (e.g. uveitis and Herpes keratitis), the kidney (e.g. renal allograft rejection and glomerulonephritis), the liver
  • liver allograft rejection e.g. liver allograft rejection, viral hepatitis, alcoholic hepatitis, cholangitis
  • the heart e.g. cardiac allograft rejection and atherosclerotic plaques
  • the bone e.g. rheumatoid arthritis
  • the thyroid e.g. Grave's disease and Hashimoto's thyroiditis
  • SUBSTTTUTE SHEET (RULE 26) (e.g. psoriasis, scleroderma, graft v host disease, contact dermatitis, lichen planus. fixed drug eruption, mycosis fungoides, and alopecia areata).
  • the term "host” refers to a human, rat, mouse, dog, cow, sheep, horse, cat and goat.
  • the present invention derives from the surprising discovery that antisense molecules which are encapsulated in a liposome or lipid particle composition can be delivered to a site of inflammation in response to overexpression of ICAM-1 and thereby reduce the associated inflammation. It was particularly surprising that liposome formulations which consist essentially of charge neutral lipids and a sterol (e.g., cholesterol) would be effective for antisense delivery in view of the conventional wisdom that cationic liposome formulations or formulations having fusogenic lipids or proteins are necessary for cell or endosome fusion.
  • a sterol e.g., cholesterol
  • compositions for the treatment of conditions associated with the overexpression of cellular adhesion molecules preferably ICAM-1.
  • These compositions comprise an antisense oligonucleotide encapsulated in a lipid mixture.
  • the lipid mixture can be in either of two forms. The first is a conventional liposome, which is preferably charge neutral, consists essentially of neutral phosphoiipids and a sterol (e.g., cholesterol) and which can be passively loaded with an antisense molecule.
  • the second form is a lipid particle which comprises phosphoiipids, cationic lipids, sterols and combinations thereof.
  • DOTMA N- ((2, 3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride
  • antisense oligonucleotides which are useful in the present invention are those oligonucleotides which are complementary to a portion of a mammalian nucleic acid encoding cellular adhesion molecules such as ELAM-1 (human), VCAM-1 (human) and ICAM-1 (human and mouse) which are provided as Sequence I.Ds. No. 1 , 3, 5 and
  • ELAM-1 is a 115-kDa membrane glycoprotein which is a member of the selecting family of membrane glycoproteins (see, Bevilacqua, et al., Science, 243: 1160- 1165 (1989)).
  • the amino terminal region of ELAM-1 contains sequences with homologies to members of lectin-like proteins, followed by a domain similar to epidermal growth factor, followed by six tandem 60-amino acid repeats similar to those found in complement receptors 1 and 2.
  • ELAM-1 is encoded by a 3.9-kb mRNA.
  • the 3 '-untranslated region of ELAM-1 mRNA contains sever sequence motifs ATTTA which are responsible for the rapid turnover of cellular mRNA consistent with the transient nature of ELAM-1 expression.
  • ELAM-1 exhibits a limited cellular distribution and has only been identified on vascular endothelial cells. Like ICAM-1, ELAM-1 is inducible by a number of cytokines including tumor necrosis factor, interleukin- 1 and lymphotoxin and bacterial lipopolysaccharide. Unlike ICAM-1, ELAM-1 is not induced by gamma- interferon. The kinetics of ELAM-1 mRNA induction and disappearance in human umbilical endothelial cells precedes the appearance and disappearance of ELAM-1 on the cell surface.
  • VCAM-1 is a 110-kDa membrane glycoprotein encoded by a 3.2-kb mRNA. It appears to be encoded by a single-copy gene which can undergo alternative splicing to yield products with either six or seven immunoglobulin domains (see Osborn, et al., Cell 59:1203-1211 (1989)).
  • the receptor for VCAM-1 is proposed to be CD29 (VLA-4) as demonstrated by monoclonal antibodies which bind to CD29 and block the adherance of Ramos cells to VCAM-1.
  • VCAM-1 is expressed primarily on vascular endothelial cells and is also regulated by treatment with cytokines (see, Rice, et al. , Science 246:1303-1306 (1989) and Rice, et al., J. Exp. Med. 171:1369-1374 (1990)).
  • ICAM-1 Human ICAM-1 is encoded by a 3.3-kb mRNA resulting in the synthesis of a 55,219 dalton protein. ICAM-1 is heavily glycosylated through N-linked
  • SUBSTTTUTE SHEET (RULE 25) glycosylation sites.
  • the mature protein has an apparent molecular mass of 90 kDa as determined by gel electrophoresis (see Staunton, et al. , Cell 52:925-933 (1988)).
  • ICAM-1 is a member of the immunoglobulin supergene family, containing 5 immunoglobulin-like domains at the amino terminus, followed by a transmembrane domain and a cytoplasmic domain. The primary binding site for LFA-1 and rhinovirus are found in the first immunoglobulin-like domain. However, the binding sites appear to be distinct.
  • ICAM-1 exhibits a broad tissue and cell distribution, and may be found on white blood cells, endothelial cells, fibroblast, keratinocytes and other epithelial cells.
  • the expression of ICAM-1 can be regulated on vascular endothelial cells, fibroblasts, keratinocytes, astrocytes and several cell lines by treatment with bacterial lipopolysaccharide and cytokines such as interleukin- 1 , tumor necrosis factor, gamma- interferon, and lymphotoxin (see, e.g., Frohman, et al., J. Neuroimmunol. 23:117-124 (1989)).
  • the antisense oligonucleotide is complementary to the 3 '-untranslated region of human ICAM-1 mRNA and contains from about 10 to about 50 nucleotides, more preferably about 15 to about 30 nucleotides.
  • the antisense oligonucleotide is a phosphorothioate oligonucleotide or a methyl phosphonate oligonucleotide.
  • Phosphorothioate oligonucleotides are those oligonucleotides in which one of the non-bridged oxygens of the intemucleotide linkage has been replaced with sulfur.
  • MeP-oligos are those oligonucleotides in which one of the non-bridged oxygens of the intemucleotide linkage has been replaced by a methyl group. These MeP-oligos have also proven to be more nuclease resistant than their natural phosphodiester linked derivatives.
  • the antisense oligonucleotides used in the present invention may be synthesized in solid phase or in solution. Generally, solid phase synthesis is preferred. Detailed descriptions of the procedures for solid phase synthesis of oligonucleotides by
  • SUBSTTTUTE SHEET (RULE 26) phosphite-triester, phosphotriester, and H-phosphonate chemistries are widely available. See, for example, Itakura, U.S. Pat. No. 4,401 ,796; Caruthers, et al. , U.S. Pat. Nos. 4,458,066 and 4.500,707; Beaucage, et al. , Tetrahedron Lett. , 22: 1859-1862 (1981); Matteucci, et al , J. Am. Chem. Soc , 103:3185-3191 (1981); Caruthers, et al .
  • timing of delivery and concentration of monomeric nucleotides utilized in a coupling cycle will not differ from the protocols typical for commercial phosphoramidites used in commercial DNA synthesizers. In these cases, one may merely add the solution containing the monomers to a receptacle on a port provided for an extra phosphoramidite on a commercial synthesizer (e.g., model 380B, Applied
  • DMT dimethoxytrityl
  • coupling efficiency may be determined by measuring the DMT cation concentration during the acidic removal of the DMT group. DMT cation concentration is usually determined by spectrophotometrically monitoring the acid wash. The acid/DMT solution is a bright orange color.
  • coupling efficiency may be estimated by comparing the ratio of truncated to full length oligonucleotides utilizing, for example, capillary electrophoresis or HPLC.
  • Solid phase oligonucleotide synthesis may be performed using a number of solid supports.
  • a suitable support is one which provides a functional group for the attachment of a protected monomer which will become the 3' terminal base in the
  • SUBSTTTUTE SHEET (RULE 26) synthesized oligonucleotide.
  • the support must be inert to the reagents utilized in the particular synthesis chemistry. Suitable supports are well known to those of skill in the art. Solid support materials include, but are not limited to polacryloylmo ⁇ holide, silica, controlled pore glass (CPG), polystyrene, polystyrene/latex, and carboxyl modified teflon. Preferred supports are amino-functionalized controlled pore glass and carboxyl- functionalized teflon.
  • Solid phase oligonucleotide synthesis requires, as a starting point, a fully protected monomer (e.g., a protected nucleoside) coupled to the solid support. This coupling is typically through the 3'-hydroxyl. Typically, a linker group is covalently bound to the 3 '-hydroxyl on one end and covalently bound to the solid support on the other end.
  • the first synthesis cycle then couples a nucleotide monomer, via its 3 '-phosphate, to the 5 '-hydroxyl of the bound nucleoside through a condensation reaction that forms a 3 '-5' phosphodiester linkage.
  • Subsequent synthesis cycles add nucleotide monomers to the 5 '-hydroxyl of the last bound nucleotide. In this manner an oligonucleotide is synthesized in a 3' to 5' direction producing a "growing" oligonucleotide with its 3' terminus attached to the solid support.
  • nucleoside monomers to a solid support
  • monomers covalently linked through a succinate or hemisuccinate to controlled pore glass are generally preferred.
  • Conventional protected nucleosides coupled through a hemisuccinate to controlled pore glass are commercially available from a number of sources (e.g. , Glen Research, Sterling, Vermont, U.S.A.; Applied Biosystems, Foster City, California, U.S.A. ; and Pharmacia LKB, Piscataway, New Jersey, U.S.A.).
  • the oligonucleotide is deprotected and cleaved from the solid support prior to use. Cleavage and deprotection may occur simultaneously or sequentially in any order. The two procedures may be interspersed so that some protecting groups are removed from the oligonucleotide before it is cleaved off the solid support and other groups are deprotected from the cleaved oligonucleotide in solution. The sequence of events depends on the particular blocking groups present, the particular linkage to a solid support, and the preferences of the individuals performing the synthesis. Where deprotection precedes cleavage, the protecting groups may be washed away from me oligonucleotide which remains bound on the solid support. Conversely, where deprotection follows cleavage, the removed
  • SUBSTTTUTE SHEET (RULE 26) protecting groups will remain in solution with the oligonucleotide. Often the oligonucleotide will require isolation from these protecting groups prior to use.
  • the protecting group on the 5 '-hydroxyl is removed at the last stage of synthesis.
  • the oligonucleotide is then cleaved off the solid support, and the remaining deprotection occurs in solution.
  • Removal of the 5 '-hydroxyl protecting group typically requires treatment with the same reagent utilized throughout the synthesis to remove the terminal 5 '-hydroxyl protecting groups prior to coupling the next nucleotide monomer.
  • deprotection can be accomplished by treatment with acetic acid, dichloroacetic acid or trichloroacetic acid.
  • oligonucleotide is a ribonucleotide and the 2 '-hydroxyl group is blocked with a tert-butyldimethylsilyl (TBDMS) moiety
  • TDMMS tert-butyldimethylsilyl
  • the latter group may be removed using tetrabutylammonium fluoride in tetrahydrofuran at the end of synthesis.
  • Phenoxyacetyl protecting groups can be removed with anhydrous ammonia in alcohol (under these conditions the TBDMS groups are stable and the oligonucleotide is not cleaved).
  • the benzoyl protecting group of cytidine is also removed with anhydrous ammonia in alcohol.
  • Cleaved and fully deprotected oligonucleotides may be used directly (after lyophilization or evaporation to remove the deprotection reagent) or they may be purified prior to use. Purification of synthetic oligonucleotides is generally desired to isolate the full lengm oligonucleotide from the protecting groups that were removed in the deprotection step and, more importantly, from the truncated oligonucleotides that were formed when oligonucleotides that failed to couple with the next nucleotide monomer were capped during synthesis. Oligonucleotide purification techniques are well known to those of skill in the art.
  • Methods include, but are not limited to, thin layer chromatography (TLC) on silica plates, gel electrophoresis, size fractionation (e.g. , using a Sephadex column), reverse phase high performance liquid chromatography (HPLC) and anion exchange chromatography (e.g., using the mono-Q column, Pharmacia-LKB, Piscataway, New Jersey, U.S.A.).
  • TLC thin layer chromatography
  • HPLC reverse phase high performance liquid chromatography
  • anion exchange chromatography e.g., using the mono-Q column, Pharmacia-LKB, Piscataway, New Jersey, U.S.A.
  • this antisense molecule acts posttranscriptionally to inhibit the expression of ICAM-1 via 2 possible mechanisms: inhibiting the formation of a stabilizing stem-loop structure necessary for mRNA stability, and by increasing the degradation of the target mRNA via increased RNase-H degradation.
  • Preparation of pharmaceutical compositions containing the antisense oligonucleotides will typically involve either encapsulating the oligonucleotide in a liposome or forming a lipid particle in which the oligonucleotide is coated with a lipid mixture.
  • the liposomes which are used in the present invention are formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phosphoiipids, preferably neutral phosphoiipids, and a sterol, such as cholesterol.
  • the selection of lipids is generally guided by consideration of, e.g. , liposome size and stability of the liposomes in the bloodstream.
  • the major lipid component in the liposomes is phosphatidylcholine.
  • Phosphatidylcholines having a variety of acyl chain groups of varying chain length and degree of saturation are available or may be isolated or synthesized by well-known techniques. In general, less saturated phosphatidylcholines are more easily sized, particularly when the liposomes must be sized below about 0.3 microns, for pu ⁇ oses of filter sterilization. Phosphatidylcholines containing saturated fatty acids with carbon chain lengths in the range of C to C 22 are preferred. Phosphatidylcholines with mono or diunsaturated fatty acids and mixtures of saturated and unsaturated fatty acids may also be used.
  • Other suitable lipids include phosphonolipids in which the fatty acids are linked to glycerol via ether linkages rather than ester linkages. Liposomes useful in the present invention may also be composed of
  • SUBSTTTUTE SHEET (RULE 26) sphingomyelin or phosphoiipids with head groups other than choline, such as ethanolamine, serine, glycerol and inositol.
  • Preferred liposomes will include a sterol. preferably cholesterol, at molar ratios of from 0.1 to 1.0 (cholesterol:phospholipid).
  • Most preferred liposome compositions are egg phosphatidylcholine/cholesterol, distearoylphosphatidy Icholine/cholesterol. dipalmitoylphosphatidy Icholine/cholesterol, and sphingomyelin/cholesterol. Methods used in sizing and filter-sterilizing liposomes are discussed below.
  • the liposomes can be prepared by any of the techniques now known or subsequently developed for preparing liposomes.
  • the liposomes can be formed by the conventional technique for preparing multilamellar lipid vesicles (MLVs), that is, by depositing one or more selected lipids on the inside walls of a suitable vessel by dissolving the lipids in chloroform and then evaporating the chloroform, and by then adding the aqueous solution which is to be encapsulated to the vessel, allowing the aqueous solution to hydrate the lipid, and swirling or vortexing the resulting lipid suspension. This process engenders a mixture including the desired liposomes.
  • MLVs multilamellar lipid vesicles
  • LUVs large unilamellar lipid vesicles
  • the lipid-containing particles can be in the form of steroidal lipid vesicles, stable plurilamellar lipid vesicles (SPLVs), monophasic vesicles (MPVs), or lipid matrix carriers (LMCs) of the types disclosed in Lenk, et al. U.S. Patent No. 4,522,803, and Fountain, et al. U.S. Patent Nos. 4,588,578 and 4,610,868, the disclosures of which are inco ⁇ orated herein by reference.
  • SPLVs stable plurilamellar lipid vesicles
  • MPVs monophasic vesicles
  • LMCs lipid matrix carriers
  • the liposomes can be subjected to multiple (five or more) freeze-thaw cycles to enhance their trapped volumes and trapping efficiencies and to provide a more uniform interlamellar distribution of solute (Mayer, et al, J. Biol. Chem. 260:802-808 (1985)).
  • the liposomes may be sized to achieve a desired size range and relatively narrow distribution of liposome sizes.
  • the liposomes may be sized to achieve a desired size range and relatively narrow distribution of liposome sizes.
  • the liposomes will have diameters of from about 50 to about 150 nm, more preferably from about 75 to about 125 nm.
  • Extrusion of liposome through a small-pore polycarbonate membrane or an asymmetric ceramic membrane is also an effective method for reducing liposome sizes to a relatively well-defined size distribution (see, U.S. Patent No. 5,008,050 and Hope, et al. , in: Liposome Technology, vol. 1, 2d ed. (G. Gregoriadis, Ed.) CRC Press, pp. 123-139 (1992), the disclosures of which are inco ⁇ orated herein by reference).
  • the suspension is cycled through the membrane one or more times until the desired liposome size distribution is achieved.
  • the liposomes may be extruded through successively smaller-pore membranes, to achieve a gradual reduction in liposome size.
  • liposomes having a size of from about 0.05 microns to about 0.15 microns are preferred.
  • Other useful sizing methods such as sonication, solvent vaporization or reverse phase evaporation are known to those of skill in the art.
  • Liposomes prepared for use in the methods and pharmaceutical compositions of the present invention may be dehydrated for longer storage.
  • the liposomes are preferably dehydrated under reduced pressure using standard freeze-drying equipment or equivalent apparatus.
  • the lipid vesicles and their surrounding medium can also be frozen in liquid nitrogen before being dehydrated or not, and placed under reduced pressure.
  • Dehydration without prior freezing takes longer than dehydration with prior freezing, but the overall process is gentler without the freezing step, and thus there is subsequently less damage to the lipid vesicles and a smaller loss of the internal contents.
  • Dehydration without prior freezing at room temperamre and at a reduced pressure provided by a vacuum pump capable of producing a pressure of about 1 mm Hg
  • SUBSTTTUTE SHEET typically takes between approximately 24 and 36 hours, while dehydration with prior freezing under the same conditions generally takes between approximately 12 and 24 hours.
  • sugars can be used, including such sugars as trehalose, maltose, sucrose, glucose, lactose, and dextran.
  • disaccharide sugars have been found to work better than monosaccharide sugars, with the disaccharide sugars trehalose and sucrose being most effective.
  • Other more complicated sugars can also be used.
  • aminoglycosides including streptomycin and dihydrostreptomycin, have been found to protect lipid vesicles during dehydration.
  • one or more sugars are included as part of either the internal or external media of the lipid vesicles.
  • the sugars are included in both the internal and external media so that they can interact with both the inside and outside surfaces of the liposomes' membranes.
  • Inclusion in the internal medium is accomplished by adding the sugar or sugars to the buffer which becomes encapsulated in the lipid vesicles during the lipid vesicle fo ⁇ nation process. Since in most cases this buffer also forms the bathing medium for the finished lipid vesicles, inclusion of the sugars in the buffer also makes them part of the external medium.
  • the amount of sugar to be used depends on the type of sugar used and the characteristics of the lipid vesicles to be protected. See, U.S. Patent No. 4,880,635 and Harrigan, et al., Chem. Phys. Lipids 52:139-149 (1990), the disclosures of which are inco ⁇ orated herein by reference. Persons skilled in the art can readily test various sugar types and concentrations to determine which combmation works best for a particular lipid vesicle preparation. In general, sugar concentrations on the order of 100 mM and above have been found necessary to achieve the highest levels of protection.
  • lipid vesicles being dehydrated are of the type which have multiple lipid layers and if the dehydration is carried to an end point where between about 2 % and about 5 % of the original water in
  • the lipid vesicles can be stored for extended periods of time until they are to be used.
  • the appropriate temperamre for storage will depend on the make up of the lipid vesicles and the temperamre sensitivity of whatever materials have been encapsulated in the lipid vesicles.
  • various oligonucleotides are heat labile, and thus dehydrated lipid vesicles containing such oligonucleotides should be stored under refrigerated conditions so that the potency of the agent is not lost.
  • the dehydration process is preferably carried out at reduced temperatures, rather than at room temperamre.
  • Methods of loading antisense oligonucleotides into liposomes will typically be carried out using an encapsulation technique in which the antisense oligonucleotide is placed into a buffer and added to a dried film of only lipid components. In this manner, the oligonucleotide will become encapsulated in the aqueous interior of the liposome.
  • the buffer which is used in the formation of the liposomes can be any biologically compatible buffer solution of, for example, isotonic saline, phosphate buffered saline, or other low ionic strength buffers.
  • the antisense oligonucleotide will be present in an amount of from about 0.01 ng/mL to about 200 mg/mL.
  • the resulting liposomes with the antisense oligonucleotide inco ⁇ orated in the aqueous interior or in the membrane are then optionally sized as described above.
  • the antisense oligonucleotide can be formulated in lipid particles such as those described in co-pending U.S. Ser. Nos. 08/484,282 and 08/485,458 each being filed on June 7, 1995 and inco ⁇ orated herein by reference.
  • Antisense lipid particles can be prepared by combining an antisense oligonucleotide with cationic lipids in a detergent solution to provide a coated antisense- lipid complex. The complex is then contacted with phosphoiipids to provide a solution of detergent, an antisense-lipid complex and phosphoiipids, and the detergent is then removed to provide a solution of serum-stable antisense-lipid particles, in which the
  • SUBSTTTUTE SHEET (RULE 26) antisense oligonucleotide is encapsulated in a lipid bilayer.
  • the particles, thus formed, have a size of about 50-150 nm.
  • serum-stable antisense-lipid particles can be formed by preparing a mixture of cationic lipids and phosphoiipids in an organic solvent; contacting an aqueous solution of the antisense oligonucleotide with the mixture of cationic and phosphoiipids to provide a clear single phase; and removing the organic solvent to provide a suspension of antisense-lipid particles, in which the antisense oligonucleotide is encapsulated in a lipid bilayer, and the particles are stable in serum and have a size of about 50-150 nm.
  • cationic lipids which are useful will include, for example, DODAC, DOTMA, DDAB, DOTAP, DC-Choi, DORI and DMRIE. These lipids and related analogs, which are also useful in the present invention, have been described in co-pending USSN 08/316,399; U.S. Patent Nos. 5,208,036, 5,264,618, 5,279,833, 5,283,185, and 5,334,761, the disclosures of which are inco ⁇ orated herein by reference. Additionally, a number of commercial preparations of cationic lipids are available and can be used in the present invention.
  • LIPOFECTIN ® commercially available cationic liposomes comprising DOTMA and DOPE, from GIBCO/BRL, Grand Island, New York, USA
  • LIPOFECTAMINE ® commercially available cationic liposomes comprising DOSPA and DOPE, from GIBCO/BRL
  • TRANSFECTAM ® commercially available cationic liposomes comprising DOGS from Promega Co ⁇ ., Madison, Wisconsin, USA.
  • An initial solution of coated antisense-lipid complexes is formed by combining the antisense oligonucleotides with the cationic lipids in a detergent solution.
  • the detergent solution is preferably an aqueous solution of a neutral detergent having a critical micelle concentration of 15-300 mM, more preferably 20-50 mM.
  • detergents include, for example, N,N'-((octanoylimino)-bis-(trimethylene))-bis- (D-gluconamide) (BIGCHAP); BRIJ 35; Deoxy-BIGCHAP; dodecylpoly (ethylene glycol) ether; Tween 20; Tween 40; Tween 60; Tween 80; Tween 85; Mega 8; Mega 9; Zwittergent ® 3-08; Zwittergent ® 3-10; Triton X-405; hexyl-, heptyl-, octyl- and nonyl- ⁇ - D-glucopyranoside; and heptylthioglucopyranoside; with octyl 0-D-glucopyranoside being the most preferred.
  • concentration of detergent in the detergent solution is typically about 100 mM to about 2 M, preferably from about 200 mM to about 1.5 M.
  • SUBSTTTUTE SHEET (RULE 26)
  • the cationic lipids and antisense oligonucleotides will typically be combined to produce a charge ratio (+/-) of about 1: 1 to about 20: 1, preferably in a ratio of about 1 : 1 to about 12:1, and more preferably in a ratio of about 2: 1 to about 6: 1.
  • the overall concentration of antisense oligos in solution will typically be from about 25 ⁇ g/mL to about 150 mg/mL, preferably from about 100 ⁇ g/mL to about 50 mg/mL, and more preferably from about 100 ⁇ g/mL to about 5 mg/mL.
  • the combination of antisense oligos and cationic lipids in detergent solution is kept, typically at room temperamre, for a period of time which is sufficient for the coated complexes to form.
  • the antisense oligos and cationic lipids can be combined in the detergent solution and warmed to temperatures of up to about 37 °C.
  • the coated complexes can be formed at lower temperatures, typically down to about 4°C.
  • the detergent solution of the coated antisense-lipid complexes is then contacted with non-cationic lipids to provide a detergent solution of antisense-lipid complexes and non-cationic lipids.
  • the non-cationic lipids which are useful in this step include, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cardiolipin, and cerebrosides.
  • the phosphoiipids are diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide or sphingomyelin.
  • the acyl groups in these lipids are preferably acyl groups derived from fatty acids having C 10 -C 24 carbon chains. More preferably the acyl groups are lauroyl, myristoyl, palmitoyl, stearoyl or oleoyl.
  • the non-cationic lipid will be 1,2-dioleoylphosphatidylethanolamine (DOPE), palmitoyl oleoyl phosphatidylcholine (POPC) or egg phosphatidylcholine (EPC).
  • DOPE 1,2-dioleoylphosphatidylethanolamine
  • POPC palmitoyl oleoyl phosphatidylcholine
  • EPC egg phosphatidylcholine
  • the antisense-lipid particles will be fusogenic particles with enhanced properties in vivo and the phospholipid will be DOPE.
  • the non-cationic lipids will further comprise polyethylene glycol-based polymers such as PEG 2000, PEG 5000 and polyethylene glycol conjugated to ceramides, as described in co-pending USSN 08/316,429, inco ⁇ orated herein by reference. .
  • the amount of phospholipid which is used in the present methods is typically about 2 to about 150 mg/mL of total lipids to about 2 to about 150 mg/mL of antisense oligonucleotide.
  • the amount of total lipid is from about 50 to about 100 mg/mL to 50 to 100 mg/mL antisense.
  • SUBSTTTUTE SHEET (RULE 26) Following formation of the detergent solution of antisense-lipid complexes and non-cationic lipids, the detergent is removed, preferably by dialysis. The removal of the detergent results in the formation of a lipid-bilayer which surrounds the antisense oligo providing serum-stable antisense-lipid particles which have a size of from about 50 nm to about 150 nm. The particles thus formed do not aggregate and are optionally sized to achieve a uniform particle size.
  • the serum-stable antisense-lipid particles can be sized by any of the methods available for sizing liposomes.
  • the sizing may be conducted in order to achieve a desired size range and relatively narrow distribution of particle sizes. Several techniques are available for sizing the particles to a desired size.
  • particles are recirculated through a standard emulsion homogenizer until selected particle sizes, typically between about 60 and 80 nm, are observed.
  • the particle size distribution can be monitored by conventional laser-beam particle size discrimination, or QELS.
  • Extrusion of the particles through a small-pore polycarbonate membrane or an asymmetric ceramic membrane is also an effective method for reducing particle sizes to a relatively well-defined size distribution.
  • the suspension is cycled through the membrane one or more times until the desired particle size distribution is achieved.
  • the particles may be extruded through successively smaller-pore membranes, to achieve a gradual reduction in size.
  • the serum-stable antisense-lipid particles can also be prepared by combining a mixture of cationic lipids and non-cationic lipids in an organic solvent; contacting an aqueous solution of the antisense oligonucleotide with the mixture of lipids to provide a clear single phase; and removing the organic solvent to provide a suspension of antisense-lipid particles, wherein the antisense oligonucleotide is encapsulated in a lipid bilayer, and the particles are stable in serum and have a size of from about 50 to about 150 nm.
  • SUBSTTTUTE SHEET (RULE 26)
  • the antisense oligos, cationic lipids and phosphoiipids which are useful in this group of embodiments are as described for the detergent dialysis preparative methods described above.
  • organic solvent which is also used as a solubilizing agent, is in an amount sufficient to provide a clear single phase mixture of antisense oligonucleotide and lipids.
  • Suitable solvents include chloroform, dichloromethane, diethylether, cyclohexane, cyclopentane, benzene, toluene, methanol, or other aliphatic alcohols such as propanol, isopropanol, butanol, tert-butanol, iso-butanol, pentanol and hexanol.
  • Combinations of two or more solvents may also be used in the present invention.
  • Contacting the antisense oligonucleotide with the organic solution of cationic and phosphoiipids is accomplished by mixing together a first solution of antisense oligonucleotide, which is typically an aqueous solution and a second organic solution of the lipids.
  • a first solution of antisense oligonucleotide which is typically an aqueous solution
  • a second organic solution of the lipids One of skill in the art will understand that this mixing can take place by any number of methods, for example by mechanical means such as by using vortex mixers.
  • the organic solvent is removed, thus forming an aqueous suspension of serum-stable antisense-lipid particles.
  • the methods used to remove the organic solvent will typically involve evaporation at reduced pressures or blowing a stream of inert gas
  • the serum-stable antisense-lipid particles thus formed will typically be sized from about 50 nm to 150 nm. To achieve further size reduction or homogeneity of size in the particles, sizing can be conducted as described above.
  • the present invention further provides methods for the treatment of pathologic conditions associated with the overexpression of ICAM-1 in a host.
  • a pharmaceutical composition as described above is administered to the host.
  • the host is a mammal, more preferably a mouse, rat, human, horse, dog, cat. cow or pig. Still more preferably, the host is human.
  • SUBSTTTUTE SHEET (RULE 26) antisense oligonucleotides, lipid mixtures and lipids are as described above for the compounds of the present invention.
  • the antisense oligonucleotide liposomes and lipid particles described above can be administered in any suitable manner, preferably with pharmaceutically acceptable carriers.
  • suitable methods of administering such compositions in the context of the present invention to an animal are available, and, although more than one route can be used to administer a particular composition, a particular route can provide a more immediate and more effective reaction than another route.
  • Pharmaceutically acceptable carriers are also well-known to those who are skilled in the art. The choice of carrier will be determined in part by the particular composition, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of the pharmaceutical composition of the present invention.
  • Formulations suitable for oral administration can consist of (a) liquid solutions, such as an effective amount of the antisense oligonucleotide (in a liposome or lipid particle) dissolved in diluents, such as water, saline or PEG 400; (b) suspensions in an appropriate liquid; and (c) suitable emulsions.
  • Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
  • the formulations can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water, for injections, immediately prior to use.
  • sterile liquid carrier for example, water
  • Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.
  • the dose administered to an animal, particularly a human, in the context of the present invention should be sufficient to effect a therapeutic response in the animal over a reasonable time frame. The dose will be determined by the strength of the particular compound employed and the condition of the animal, as well as the body
  • SUBSTTTUTE SHEET (RULE 26) weight or surface area of the animal to be treated.
  • the size of the dose also will be determined by the existence, nature, and extent of any adverse side-effects that might accompany the administration of a particular compound in a particular animal.
  • the antisense oligonucleotides in liposome or lipid particle formulations
  • compositions of the present invention can be administered at the rate up to 3000 mg/m 2 body surface area, which approximates 6 grams/day in the average patient.
  • a preferred rate is from 1 to 300 mg/m 2 body surface area. This can be accomplished via single or divided doses.
  • such compounds can be administered at the rate of up to about 2500 mg/m 2 /d, preferably from about 0.1 to about 200 mg/m 2 /d.
  • such compounds can be administered at the rate of up to about 2500 mg/ ⁇ r/d, preferably from about 0.1 to about 200 mg/m 2 /d.
  • the rate can be up to about 2500 mg/m 2 /d, preferably from about 0.1 to about 200 mg/m 2 /d.
  • the dose for inhalation aerosol administration can be up to about 2500 mg/m 2 /d, preferably from about 0.1 to about 200 mg/m 2 /d.
  • Direct intraperitoneal administration can be performed using up to about 3000 mg/m 2 /d, preferably from about 0.1 to about 100 mg/m 2 /d.
  • the dose for reservoir administration to the brain or spinal fluid can be up to about 2000 mg/m 2 /d, preferably from about 0.1 to about 100 mg/n /d.
  • the dose can be from about 0.1 to about 5000 mg/day in a bolus, preferably from about 1.0 to about 200 mg/day.
  • the dose can be up to about 2000 mg/m 2 /d, preferably from about 0.1 to about 100 mg/m 2 /d.
  • DSPC disearoyl-sn-glycero-3-phosphocholine
  • EPC egg phosphatidylcholine
  • VBS 5X Veronal Buffered Saline
  • GVB 2 "GVB 2" .
  • Store at 4°C. Make this buffer every few days.
  • DGVB 2+ Mix 250 mL of D5W with 250 mL of GVB 2 . Add 75 ⁇ L of 1.0 M CaCl 2 and 100 ⁇ L of 2.5 M MgCl 2 . Store at 4°C. Make this buffer every few days.
  • EDTA EDTA
  • GVB 2 distilled water
  • This example illustrates the passive encapsulation of antisense oligonucleotides in liposomes. Additionally, this example illustrates that liposomes and unencapsulated DNA can be separated by their molecular weight difference using size exclusion chromatography .
  • EPC Egg phosphatidylcholine
  • CH cholesterol
  • ISIS 2302 The antisense molecule ISIS 2302 (1.5 g) was dissolved in 10 mL of sterile phosphate buffered saline (PBS) and added to the lipid film composed of 2.5 g of EPC:CH (55:45; mol: mol).
  • the resulting multilamellar vesicles were transferred to three 5 mL cryovials and subjected to five cycles of freezing in liquid nitrogen and thawing at 40 °C.
  • the freeze-thawed MLV were combined, transferred into a sterile 100 mL capacity extruder (Lipex Biomembranes, Vancouver) and extruded ten times through one 0.1 ⁇ m filter.
  • Non-encapsulated (free) antisense molecule was separated from the entrapped molecule by anion exchange chromatography using DEAE-Sepharose CL-6B.
  • the columns were washed with sterile PBS followed by elution of 300 mL of 100 nm vesicles composed of EPC (24 mg/mL in PBS).
  • Columns consisted of 6 mL syringes with a packed volume of 5 mL. A 400 mL aliquot of the antisense-liposome suspension was eluted on each column and the fractions containing lipid were collected and combined. Each column was used twice and then stored at 4°C for future recovery of the free antisense molecule.
  • the total volume of the column eluant was approximately 110 mL; therefore, it was necessary to concentrate the liposomal antisense.
  • the liposomal antisense was placed in dialysis tubing (MW cutoff, 14000) and concentrated to approximately 28 mL using Aquacide. Results of this purification are shown in Figure 2.
  • Trapping efficiency was determined by size exclusion chromatography using a 1 mL Biogel A 15m (fine) spin column as described previously (Chonn et al ,
  • DNA in 250 ⁇ L H 2 O was separated from the lipid by the addition of 750 ⁇ L of chloroform: methanol (1 :2.1) to form a single phase consisting of chloroform:methanol:H 2 O (1:2.1:1). Additional volumes of H 2 O (250 ⁇ L) and chloroform (250 ⁇ L) were added to the sample, resulting in a two phase system. The sample was then centrifuged at 3000 ⁇ m for 10 min to facilitate rapid separation of the organic and aqueous layers. The upper, aqueous, phase was collected and 400 ⁇ L was assayed for DNA by absorbance at 260 nm.
  • the lower, chloroform, phase was washed three times with 300 ⁇ L of methanol :H 2 0 (1: 1) and dried under N 2 .
  • the lipid film was hydrated in 1 mL of H 2 0 and an aliquot was taken for phosphate assay.
  • liposomal antisense preparation was not always used immediately it was necessary to analyze the stability of the preparation over several days. To this end, retention of antisense in liposomes was measured over a 5 day period, at 4°C and at room temperamre. Leakage of entrapped antisense would appear as a peak in the included volume of Biogel A15m spin columns. At various times, 50 ⁇ L aliquots of the sample were applied to a 1 mL Biogel A15m column and eluted as described above. Lipid and oligonucleotide were determined as described above. A second peak in the oligonucleotide elution profile was taken to indicate leakage of entrapped oligonucleotide.
  • This example illustrates the complement activation by a liposomal antisense formulation.
  • the assay is a two-step procedure. The first step involves consumption of complement by liposomes and/or DNA, while the second step involves the lysis of antibody-sensitized sheep red cells by any residual complement that may not have been activated in the first part of the assay.
  • the first component of the assay that was tested was the activity of the fresh serum pool. This should be tested each time a new serum pool is generated as there will be some differences in complement activity between serum pools which can affect the sensitivity of the assay.
  • a series of serum dilutions was tested to determine what dilution would give both maximal red cell lysis and minimal interference in absorbance readings (the more concentrated the serum dilution the more background absorbance is observed). Anything less than a 100-fold serum dilution gave reasonable levels of red cell lysis.
  • EDTA-GVB 2' was added at the end of the assay to inhibit complement activity. The volume of EDTA-GVB 2" can be modified to increase or decrease the absorbance range of the assay depending on the activity of the semm pool.
  • SUBSTTTUTE SHEET (RULE 26) Blood from seven healthy males and six healthy females was gathered into chilled serum mbes and immediately placed in an ice/ water bath. Thirty mL of blood was collected per individual. Tubes were centrifuged at 2500 ⁇ m for 10 min at 4°C, every six mbes (to avoid clotting). Plasma was removed from all mbes and pooled into a 250 mL beaker, on ice. The pooled plasma was then incubated at 37°C for 30 min, in the presence of several cloning sticks (to help recess the clot). The clot was removed and recessed, generating approximately 100 mL of serum. The semm was aliquoted (1.0 mL) into 1.5 mL Eppendorf mbes and stored at -65°C until use.
  • the cell suspension was warmed to 37 °C in a shaking bath and rabbit anti-sheep red blood cell antibody (hemolysin) was added to give a final antibody dilution of 1/500 (i.e. 20 ⁇ L of antibody into 10 mL of cells). This mixmre was incubated for 30 min at 37°C. Following the. incubation, the cells were centrifuged at 1500 ⁇ m for 5 min at 4°C, the supernatant removed, and the cells washed with EDTA-GVB 2' . The cells were then washed 2 times with DGVB 2+ in order to further remove any free antibody and to introduce cations into the cell suspension. Finally, the cell concentration was adjusted to
  • SUBSTTTUTE SHEET (RULE 26) 2 x 10 a cells/mL with DGVB 2+ using the information given above. Cells were maintained at 4°C at all times, after preparation, and were used on the same day.
  • the mixmre was incubated for 30 min at 37 °C and subsequently placed on ice.
  • EDTA-GVB 2 (1.0 mL) was added to the sample to inhibit complement activity and the mixmre was centrifuged for 5 min at 4°C and 1500 ⁇ m. Aliquots of the supernatant (250 ⁇ L) were transferred to a microtiter plate, in triplicate, with care not to disturb the pelleted red cells. The absorbance of the supernatant was measured at 410 nm on an electronic plate reader.
  • Figure 5 depicts the complement activating ability of the liposome composition. As can be seen, the neutral EPC:CH liposomes showed no observable complement activation over the concentration ranges studied.
  • DTH delayed type hypersensitivity
  • SUBSTTTUTE SHEET (RULE 26) edema (ear thickness measurements), vascular leak (liposome accumulation in inflamed ears), and cell infiltration (myeloperoxidase assays for neutrophils/monocytes or by prelabeling bone marrow cells and circulating leukocytes with [ 3 H] -thymidine). Furthermore, both inbred (BALB/c) and outbred (ICR) mice have been tested in this model, with similar patterns of inflammation being observed for both strains of mice.
  • mice Female BALB/c and ICR mice were obtained from Harley and Sprague Davis. BALB/c mice were used at 6-9 weeks of age, while ICR mice were used at 8-10 weeks of age. Each experimental group consists of four mice and the experiments were repeated at least twice.
  • mice were sensitized by applying 25 ⁇ L of 0.5% 2,4-dinitro-l- fluorobenzene (DNFB) in acetone: olive oil (4: 1) to the shaved abdominal wall for two consecutive days. Four days after the second application, mice were challenged on the dorsal surface of the left ear with 10 ⁇ L of 0.2% DNFB in acetone:olive oil (4: 1). Mice received no treatment on the contralateral (right) ear. In some cases, control mice received 10 ⁇ L of vehicle on the dorsal surface of the left ear.
  • DNFB 2,4-dinitro-l- fluorobenzene
  • Ear thickness was measured immediately prior to ear challenge, and at various time intervals after DNFB challenge, using an engineer's micrometer (Mitutoyo, Tokyo, Japan). Increases in ear thickness measurements were determined by subtracting the pre-challenge from post-challenge measurements.
  • mice demonstrate peak ear thickness measurements between 24-48 hours after ear challenge.
  • the maximal ear thickness measurements exhibited by these mice were 130 x 10" inches, which corresponds to an increase of -50% over baseline values (75-85 x 10" inches).
  • DSPC distearylphosphatidylcholine
  • CH cholesterol
  • Liposomes contained a non-exchangeable radioactive lipid marker, [ 3 H]cholesterylhexadecylether (CHE).
  • LUVs were administered at a dose of 100 mg/kg (200 ⁇ L; - 2 ⁇ Ci of CHE/mouse) via the dorsal tail vein at 0, 24, 48, 72 hr after initiation of ear inflammation. Liposomes were allowed to circulate for 24 hr after taking ear measurements. Mice were then terminated and the ears were collected for analysis of liposome accumulation and cell infiltration.
  • the second portion was freeze-thawed five times in liquid nitrogen and again sonicated for 30 sec (power output, 4; 40% pulse). The sample was then centrifuged for 10 min at 18 000 x g to remove cellular debris. The supernatant was removed and assayed for MPO activity by incubating 0.1 mL aliquots with 2.9 mL of substrate buffer (50 mM potassium phosphate, pH 6.0 containing 0.167 mg/mL o- dianisidine dihydrochloride and 0.0005% hydrogen peroxide). Absorbance at 460 nm was monitored for several minutes and, in most cases, was taken from the absorbances at 30 and 90 seconds. All solutions were maintained on ice as much as possible through the procedure.
  • One unit of MPO activity is defined as the amount of enzyme that degrades 1 ⁇ mol of peroxide per minute at 25 °C.
  • Peak MPO activity occurs at approximately 48 hr and returns to baseline levels by 96 hr. Based on the accumulation of radiolabeled blood cells (predominantly neutrophils, monocytes, and T-lymphocytes), cell accumulation peaks by 24 hr and remains relatively high over 72 hours (see Figure 11). The major difference in these two procedures is that the MPO assay primarily measures neutrophil accumulation (neutrophils have three times more MPO than monocytes), whereas the [ 3 H] -methyl thymidine procedure measures the influx of all cells equally. Neutrophil accumulation at sites of inflammation has been demonstrated to rise rapidly over the first 24 hours and to decrease almost as rapidly. From 24-48 hours, increased levels of monocytes and T-cells are observed at the inflammation site.
  • This example illustrates the passive targeting of large unilamellar vesicles to sites of inflammation using a murine ear inflammation model.
  • Liposome accumulation appeared to be maximal during the 0-24 and 24-48 hr time periods after the onset of inflammation, corresponding to peak inflammatory events. After this, liposome accumulation decreased dramatically, corresponding to remodeling and repair of the "leaky" vasculature.
  • inflainmation are the mo ⁇ hological and functional alterations that occur in dermal microvascular cells. When activated by cytokines, endothelial cells vasodilate, resulting in increased vascular blood flow to the region of inflammation. In addition, the blood vessels are stimulated to structurally remodel, thus enabling immune cells to extravasate from the vasculamre and access the inflammation site.
  • the endothelium is optimized for the infiltration of leukocytes and macromolecules to the site of inflammation. Consequently, it would be expected that relatively small vesicles, such as liposomes (100 nm diameter in our smdies), would avidly move through the "leaky” vasculamre and passively accumulate at sites of inflammation.
  • mice used, as well as the sensitization and elicitation of contact sensitivity were carried out as described above in Example 3. Liposome accumulation was monitored over the first 24 hours of inflammation. LUVs were administered at a dose of 100 mg/kg (200 ⁇ L; - 2 ⁇ Ci of CHE/mouse) via the dorsal tail vein immediately
  • Liposome accumulation was examined during various stages of murine ear inflammation so as to give a relative indication of the ability of these vesicles to extravasate through the "inflamed" vasculamre. This is of interest for the passive targeting of liposomal dmgs, such as anti-ICAM-1 oligonucleotides and corticosteroids, to sites of inflammation. Liposome accumulation was measured over the following time intervals: 0-24 hr, 24-48 hr, 48-72 hr. For both ICR and BALB/c mice, maximal liposome accumulation occurred during the first 24 hr of inflammation. Thus, it appears that the most prominent stmcmral changes to the vasculamre occurred during the 0-24 hr time period. This is consistent with previous reports detailing vascular leakage of relatively small molecules and proteins, such as BSA.
  • lipid steadily accumulated in the inflamed ear during the first 24 hr, corresponding to the remodeling of the vascular endothelium in the inflamed region. No such increases in lipid accumulation were observed for control ears (non- inflamed), since no remodeling of the endothelium has occurred.
  • This example illustrates the plasma clearance and biodistribution of free and encapsulated oligonucleotides.
  • SUBSTTTUTE SHEET (RULE 26) Mice were sensitized and challenged as described in Example 3. Fifteen minutes after ear challenge, various antisense formulations were administered by the lateral tail (200 ⁇ L) at an oligonucleotide dose of 50 mg/kg. Control mice were injected with PBS or saline. At various timepoints blood was withdrawn from the mice by cardiac puncture and collected into plasma mbes containing EDTA. An aliquot of whole blood was removed for analysis. The blood was centrifuged at 3000 ⁇ m for 10 min and an aliquot of the plasma was counted by standard liquid scintillation analysis.
  • Figure 15 shows the tissue distribution of pH] -antisense in the liver, spleen, lung, and kidney after intravenous injection of oligonucleotide (50 mg/kg dose).
  • the antisense molecule was rapidly cleared from the plasma with the majority of the dose distributing primarily to the liver and kidney. Only minor accumulation was observed in the spleen and lung. On the basis of organ weight, however, the kidney was the most efficient organ for antisense removal. This result is consistent with reports that indicate that the major route of elimination of free antisense from the body is by urinary excretion.
  • Figure 16 demonstrates the biodistribution profiles for both the lipid and antisense portions of the encapsulated formulation. The liver was found to be the primary organ of accumulation for both antisense and lipid.
  • biodistribution of the lipid component determines the distribution of the antisense molecule as would be expected if the antisense molecule does not leak out of the liposomes.
  • This example illustrates the efficacy of mouse anti-ICAM oligonucleotide.
  • the efficacy of antisense oligonucleotide against mouse ICAM-1 mRNA was tested using the ear inflammation model described above (Example 3).
  • the test oligonucleotide was developed by Isis Pharmaceuticals and is referred to as Isis 3082.
  • the antisense is a 20 base (20 mer) phosphorothioate against a sequence in the untranslated 5 '-region of murine ICAM-1 mRNA (see, Bennett, et al., J. Immunol. 152:3530-3540 (1994); Bennett, et al., Adv.
  • Ear thickness in the mice was measured immediately prior to ear challenge and 24 hr after DNFB challenge using an engineer's micrometer (Mitutoyo, Tokyo,
  • Encapsulated antisense oligonucleotides were injected as described above and the results are shown in Figure 18. Increases in ear thickness were observed in mice
  • ISIS 3082 an encapsulated human ICAM-1 specific oligonucleotide encapsulated in EPC:CH liposomes was able to significantly reduce ear edema, whether injected 30 min prior to or immediately after initiating inflammation. This result is comparable to the control in which mice were treated topically with corticosteroid (HBP).
  • GCA ATT CAA AAC AAA
  • GAA GAG ATT GAG TAC CTA
  • AAC TCC ATA TTG AGC 308 Ala He Gin Asn Lys Glu Glu He Glu Tyr Leu Asn Ser He Leu Ser 50 55 60
  • AGT CCA CTG AAT GGG AAG GTG ACG AAT GAG GGG ACC ACA TCT ACG CTG 240 Ser Pro Leu Asn Gly Lys Val Thr Asn Glu Gly Thr Thr Ser Thr Leu 65 70 75 80
  • AGA AAA GCC AAC ATG AAG GGG TCA TAT AGT CTT GTA GAA GCA CAG AAA 2208 Arg Lys Ala Asn Met Lys Gly Ser Tyr Ser Leu Val Glu Ala Gin Lys 725 730 735
  • GAG ACC CCG TTG CCT AAA AAG GAG TTG CTC CTG CCT GGG AAC AAC CGG 240 Glu Thr Pro Leu Pro Lys Lys Glu Leu Leu Leu Pro Gly Asn Asn Arg 65 70 75
  • GAG TTC GAC AGA ACC CTG CCG CTG CGC TGC GTT TTG GAG CTA GCG GAC 916 Glu Phe Asp Arg Thr Leu Pro Leu Arg Cys Val Leu Glu Leu Ala Asp 285 290 295
  • AAC CCA TCT CCT AAA ATG ACC TGC AGA CGG AAG GCA GAT GGT GCC CTG 1348 Asn Pro Ser Pro Lys Met Thr Cys Arg Arg Lys Ala Asp Gly Ala Leu 430 435 440
  • AAACGCTGAC TTCATTCTCT ATTGCCCCTG CTGAGGGGCT CCTGCCTAAG GAAGACATGA 1933

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Abstract

La présente invention se rapporte à des compositions pharmaceutiques destinées au traitement des troubles pathologiques associés à la sur-expression de la molécule-1 d'adhésion intercellulaire (ICAM-1) chez un hôte. Ces compositions pharmaceutiques comportent une quantité efficace d'une molécule anti-sens dirigée contre l'expression d'ICAM-1 et encapsulée dans un mélange lipidique qui est typiquement un liposome ou une particule lipidique. Ledit mélange lipidique doit comporter spécifiquement au moins deux éléments sélectionnés dans le groupe constitué par des phospholipides, des stérols et des lipides cationiques.
PCT/CA1997/000347 1996-05-30 1997-05-22 Efficacite amelioree d'apport liposomal de molecules anti-sens WO1997046671A1 (fr)

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JP10500030A JP2000511541A (ja) 1996-05-30 1997-05-22 リポソームアンチセンス送達の効果の増強
EP97921565A EP0906421A1 (fr) 1996-05-30 1997-05-22 Efficacite amelioree d'apport liposomal de molecules anti-sens

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WO1998051278A2 (fr) 1997-05-14 1998-11-19 Inex Pharmaceuticals Corporation Encapsulation hautement efficace d'agents therapeutiques charges dans des vesicules lipidiques
JP2001316297A (ja) * 2000-02-23 2001-11-13 Kaken Pharmaceut Co Ltd 遺伝子包埋リポソーム製剤及びその製法
US6670321B1 (en) * 1998-12-30 2003-12-30 The Children's Medical Center Corporation Prevention and treatment for retinal ischemia and edema
US6835395B1 (en) 1997-05-14 2004-12-28 The University Of British Columbia Composition containing small multilamellar oligodeoxynucleotide-containing lipid vesicles
WO2006081331A2 (fr) 2005-01-25 2006-08-03 Prolexys Pharmaceuticals, Inc. Erastine et proteines de liaison d'erastine, et utilisations de celles-ci
EP2113247A2 (fr) 1997-05-14 2009-11-04 The University Of British Columbia Encapsulation hautement éfficace d'acides nucléiques dans des vésicules lipidiques
US20210332386A1 (en) * 2018-10-19 2021-10-28 Ohio State Innovation Foundation Extracellular vesicles for targeted therapies against myeloid-derived suppressor cells

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WO1994022468A1 (fr) * 1993-04-02 1994-10-13 Anticancer, Inc. Procede permettant l'apport aux follicules pileux de compositions profitables
WO1996010390A1 (fr) * 1994-09-30 1996-04-11 Inex Pharmaceuticals Corp. Nouvelles compositions d'introduction de substances polyanioniques dans des cellules

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WO1994005333A1 (fr) * 1992-09-02 1994-03-17 Isis Pharmaceuticals, Inc. Modulation oligonucleotidique de l'adhesion cellulaire
WO1994022468A1 (fr) * 1993-04-02 1994-10-13 Anticancer, Inc. Procede permettant l'apport aux follicules pileux de compositions profitables
WO1996010390A1 (fr) * 1994-09-30 1996-04-11 Inex Pharmaceuticals Corp. Nouvelles compositions d'introduction de substances polyanioniques dans des cellules

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STEPKOWSKI, S. ET AL.: "Blocking of heart allograft rejection by intercellular adhesion molecule-1 antisense oligonucleotides alone or in combination with other immunosuppressive modalities", JOURNAL OF IMMUNOLOGY, vol. 153, 1994, BALTIMORE US, pages 5336 - 5346, XP002040367 *
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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998051278A2 (fr) 1997-05-14 1998-11-19 Inex Pharmaceuticals Corporation Encapsulation hautement efficace d'agents therapeutiques charges dans des vesicules lipidiques
US6287591B1 (en) 1997-05-14 2001-09-11 Inex Pharmaceuticals Corp. Charged therapeutic agents encapsulated in lipid particles containing four lipid components
US6835395B1 (en) 1997-05-14 2004-12-28 The University Of British Columbia Composition containing small multilamellar oligodeoxynucleotide-containing lipid vesicles
US6858225B2 (en) 1997-05-14 2005-02-22 Inex Pharmaceuticals Corporation Lipid-encapsulated polyanionic nucleic acid
EP2113247A2 (fr) 1997-05-14 2009-11-04 The University Of British Columbia Encapsulation hautement éfficace d'acides nucléiques dans des vésicules lipidiques
US6670321B1 (en) * 1998-12-30 2003-12-30 The Children's Medical Center Corporation Prevention and treatment for retinal ischemia and edema
JP2001316297A (ja) * 2000-02-23 2001-11-13 Kaken Pharmaceut Co Ltd 遺伝子包埋リポソーム製剤及びその製法
WO2006081331A2 (fr) 2005-01-25 2006-08-03 Prolexys Pharmaceuticals, Inc. Erastine et proteines de liaison d'erastine, et utilisations de celles-ci
US20210332386A1 (en) * 2018-10-19 2021-10-28 Ohio State Innovation Foundation Extracellular vesicles for targeted therapies against myeloid-derived suppressor cells

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