EP0950060A1 - Inhibition antisens de molecules d'adhesion humaines - Google Patents

Inhibition antisens de molecules d'adhesion humaines

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Publication number
EP0950060A1
EP0950060A1 EP96945380A EP96945380A EP0950060A1 EP 0950060 A1 EP0950060 A1 EP 0950060A1 EP 96945380 A EP96945380 A EP 96945380A EP 96945380 A EP96945380 A EP 96945380A EP 0950060 A1 EP0950060 A1 EP 0950060A1
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Prior art keywords
oligonucleotide
selectin
vcam
icam
human
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German (de)
English (en)
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EP0950060A4 (fr
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Glenn D. Hoke
Matthews O. Bradley
Taffy J. Williams
Che-Hung Lee
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Dyad Pharmaceutical Corp
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Dyad Pharmaceutical Corp
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Publication of EP0950060A1 publication Critical patent/EP0950060A1/fr
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Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates

Definitions

  • This invention relates to the use of specific antisense oligonucleotides to inhibit the expression of proteins that mediate cell to cell association. Particularly, this invention relates to the inhibition of cellular adhesion molecules through the application of antisense oligonucleotides. More specifically, this invention relates to the use of certain sequence specific antisense oligonucleotides complementary to human mRNAs or pre-mRNAs coding for Intercellular Adhesion Molecule-1, ICAM-1, Endothelial Leukocyte Adhesion Molecule-1, E-selectin, and Vascular Cell Adhesion ⁇ Molecule-1, VCAM-I. Most specifically, this invention relates to the use of antisense oligonucleotides target in human ICAM-1, E-selectin, or VCAM-1 for treating or preventing human diseases or conditions related to inflammation.
  • Inflammation is characterized by the local accumulation of leukocytes, plasma proteins, and fluid usually at an extravascular site. Inflammatory processes are intrinsically destructive to the surrounding tissues and may, in certain circumstances such as allograft rejection or sepsis, be more harmful than beneficial. Thus, an appropriate strategy for evaluating conditions associated with an inflammatory component is down-regulation, but not total ablation, of the inflammatory response. Down-regulation of specific cell adhesion receptors and/or ligands to the receptors will prevent or lessen the inflammatory mediated damage to endothelial cells in the vasculature.
  • leukocytes The migration of leukocytes into tissues is the central event in the immune or inflammation response. This migration to and subsequent emigration into the tissue is responsible for the successful host response to injury and infection.
  • the leukocytes are also potentially harmful and contribute to the pathology of many inflammatory disorders.
  • the precise mechanism of this injury is not known, but the generation of free oxygen radicals and release of proteolytic enzymes have been implicated and may act together in leukocyte induced endothelial cell damage (Varani, J. et al. (1989), Am. J. Path. 135: 429-436 " ).
  • Evidence for the leukocyte adhesion to endothelial cells has been attributed to specific surface proteins.
  • Adhesion molecules are activated by various cellular mediators, exogenous or endogenous to the host, and therefore, the logical approach is down-regulation of adhesion protein expression as opposed to treatments aimed at the multiple activators.
  • antisense oligonucleotides to specifically down-regulate adhesion protein expression offers obvious advantages to evaluating their role in inflammation.
  • a family of endothelial cell adhesion molecules known collectively as the immunoglobin superfamily are involved in the binding of leukocytes to activated endothelium.
  • ICAM-1 intercellular cell adhesion molecule-1
  • ICAM-1 intercellular cell adhesion molecule-1
  • CAM-1 is a 90 kD cell-surface glycoprotein of endothelial cells that binds neutrophils and perhaps monocytes via the Lymphocyte Function Associated- 1, LFA-1, integrin expressed by white blood cells (Kishimoto, T.K. et al. (1989) Adv. Immunol. 46: 149-182).
  • ICAM-1 interleukins, i.e. interleukin-1, and tumor necrosis factor-alpha, TNF- ⁇ (Prober, J.S. and R.S. Cotran (1990) Transplant. 50: 537-544).
  • ICAM-1 The ability of antibodies to ICAM-1 to block the adhesion of neutrophils, eosinophils, and basophils induced in vitro in HUVECs by interleukin-1 suggests that ICAM-1 plays an important role during the inflammatory response (Bochner, B.S. et al. (1991) J. Exp. Med. 173: 1553-1556 and Carlos, T. et al. (1991) Blood 77: 2266-2271).
  • the ability to target the pre- or mature-mRNA with antisense oligonucleotides with the express purpose of down-regulating the synthesis of ICAM-1 is immediately possible.
  • the mRNA coding for ICAM-1 has been cloned and the nucleic acid sequence is available for selective targeting with antisense oligonucleotides (Staunton, D.E. et al., Cell 52-925-933; Wawryk, S.D. et al., Intl. Immunol 3: 83-93) .
  • Evidence for the leukocyte adhesion to endothelial cells has been attributed to specific surface proteins.
  • E-selectin is a 110 kD cell-surface glycoprotein of endothelial cells that binds neutrophils, and perhaps monocytes (Bevilacqua, M.P., et al., 1987, Proc. Natl. Acad. Sci. USA 84: 9238-9242).
  • E-selectin may be involved in the second, or delayed, component of leukocyte adhesion (Bevilacqua, J.S., et al., 1987, Proc. Natl. Acad. Sci. USA 84: 9238-9242 and Bevilacqua, J.S. et al, 1989, Science 243: 116-1165).
  • mediators of an inflammatory response can increase the adhesion of leukocytes to endothelial cells through the biosynthesis and expression of E-selectin.
  • HUVECs Stimulation of HUVECs with either IL-1 or TNF- ⁇ results in more than a 100-fold increase in the surface presentation of E-selectin (Osborn, L. et al., 1990, Cell 62: 3-6). Also, the stimulation of E-selectin synthesis and its presentation on the surface of HUVECs has been shown to be mediated by endotoxin (Munro, J.M. et al., 1991, Lab. Invest. 64: 295-299). Injection of endotoxin into the skin of baboons results in the strong, widespread endothelial binding of anti-E-selectin antibodies in the venules within 2 hours of injection.
  • E-selectin cDNA and genomic clones have been isolated and the nucleic acid sequence of the pre- and mature mRNA can be determined from these sequences (Goelz, S.E. et al., 1990, Cell 63: 1349-1355; Hession, C. et al., 1990, Proc. Natl. Acad. Sci. USA 87: 1673-1677; and Collins, T. et al., 1991, J. Biol. Chem. 266: 2466-2473) .
  • ICAM-1 is the ligand for the leukocyte integrin receptor molecule, LFA-1, however, antibodies against LFA-1 do not block the adhesion of lymphocytic cells to activated endothelial cells (Haskard, D., et al. (1986) J. Immunol. 137: 2901-2906). These results suggest another pathway for lymphocyte adhesion to activated endothelium.
  • VCAM-1 was identified in activated human umbilical endothelial cells by expression cloning (Osborne, L., et al. (1989) Cell 59: 1203-1211) and specific monoclonal antibodies (Rice, G.E. and M.P. Bevilacqua (1989) Science 246: 1301-1306).
  • VCAM-1 is an inducible endothelial cell surface molecule which has been demonstrated to mediate intercellular adhesion via interaction with the integrin VLA-4, which is expressed on monocytes, lymphocytes, basophils, eosinophils, and certain tumor cells, but not neutrophils (Elices, M.J., et al. (1990) Cell 60: 577-584; Taichman, D.B., et al. (1991) Cell Regul. 2: 347-356; Bochner, B.S., et al. (1991) J. Exp. med. 173: 1553-1556; Rice G.E., et al. (1990) J. Exp. Med. 171: 1369-1374) .
  • VCAM-1 expression is inducible on vascular endothelium in pathological conditions, however, it is constitutively expressed on some nonvascular cells (Rice, G.E., et al. (1990) J. Exp. Med. 171: 1369-1374; Rice, G.E., et al. (1991) AM. J. Pathol. 138: 385-393) .
  • VCAM-1 is up-regulated at the level of translation on endothelial cells of the post-capillary venules (Briscoe, D.M., et al. (1991) Transplantation 51: 537-547).
  • VCAM-1 is a transmembrane protein and a member of the immunoglobin gene superfamily.
  • VCAM-1 The VCAM-1 molecule comprises six immunoglobin-like domains which interact with the VLA-4 receptor on lymphocytes.
  • VCAM-1 expression is rapidly induced by IL-1 and TNF- ⁇ and this induction is sustained for up to 72 hours. This time course of VCAM-1 induction parallels the sustained mononuclear lymphocytic infiltration that occurs in delayed hypertension reactions (Dvorak, H.F., et al. (1980) Int. Rev. Exp. Pathol. 21: 195-199).
  • HUVECs express ICAM-1 and E-selectin upon exposure to cytokines and their expression occurs at sites of cytokine injection in vivo (Cotran, R.S. and Pobar, J.S. (1988) In Endothelial Cell Biology, N. Simionescu and M. Simionescu, eds.(New York: Plenum Press), pp. 335-347). HUVECs also express VCAM-1 upon exposure to cytokines (Osborne, L., et al. (1989) Cell 59: 1203-1211) . 3) Frozen sections of human synovium exhibit the capacity to bind lymphocytes to inflamed vessels and not normal vessels, consistent with the presence of inducible VCAM-1 at these sites.
  • VCAM-1 may be the central mediator of lymphocyte recruitment to the sites of inflammation in vivo. While the role of VCAM-1 in the adherence of resting T-cells to interleukin-1 stimulated endothelial cells has been suggested, there is little evidence that VCAM-1 is involved in the binding of either activated T-cells or in the transendothelial migration of T-cells (Oppenheimer-Marks, N., et al. (1991) J. Immunol. 147: 2913-2921) . Also, VCAM-1 does not appear to play a vital role in the adherence of activated T-cells to endothelial cells.
  • the interaction of the antisense oligonucleotide with its target mRNA is highly specific as hybridization is determined by the sequence of bases complementary to the antisense oligonucleotide, or by the Watson/Crick base pairing of the two strands of nucleic acid.
  • the specificity derived from the Watson/Crick base pairing is not evident in traditional drugs that inhibit the activity of proteins or mimic their action.
  • the side effects resulting from the use of drug therapies occur through interactions at a few contact points between the drug and various proteins that possess similar binding sites or sites of interaction. Such adverse effects will be eliminated with antisense drugs.
  • duplex stability is determined by many factors, of which length is of high importance, there are many oligonucleotides shorter than 15 nucleotides that may possess the capacity to form sufficiently stable heteroduplexes with mRNA.
  • oligonucleotides shorter than 15 nucleotides that may possess the capacity to form sufficiently stable heteroduplexes with mRNA.
  • thermodynamics approach of Breslauer et al (1986) Proc. Natl. Acad. Sci. 83: 3746-3750 and Freier, et al (1986) Proc. Natl. Acad. Sci. 83: 9373-9377
  • PO-ODN phosphodiester deoxyoligonucleotides
  • PS-ODN phosphorothioate deoxyoligonucleotide
  • antisense PS-ODNs are capable of down-regulating the in vitro expression of one member of the immunoglobin superfamily adhesion proteins involved in cell-to-cell adhesion, ICAM-1 (Chiang, M-Y. et al., J. Biol. Chem 266-18162-18171, 1991) .
  • ICAM-1 immunoglobin superfamily adhesion proteins involved in cell-to-cell adhesion, ICAM-1
  • antisense oligonucleotides in the down-regulation of ICAM-1 and other adhesion molecules should provide a basis for therapy and treatment of inflammatory disorders. Inhibition of the inflammatory response component may provide the necessary protection to prevent the deleterious consequences brought about by the body's own immune system.
  • an object of the present invention is the identification of antisense oligonucleotides that possess the capacity to inhibit the synthesis of human ICAM-1, E-selectin and VCAM-1.
  • An additional object of this invention is to provide an approach to evaluate the role these molecules play in the inflammatory process by inhibiting the expression of these proteins in vitro and in vivo using antisense oligonucleotides.
  • the foregoing objects of the present invention are accomplished by providing antisense oligonucleotides that inhibit the expression of ICAM-1, E-selectin, and VCAM-1.
  • antisense oligonucleotides is used to mean any natural or modified oligonucleotide or chemical entity that binds specifically to a pre-mRNA or mature mRNA which results in interference or inhibition with translation of the mature mRNA or prevents the synthesis of the polypeptide encoded by the mature mRNA.
  • IMM-1 Intercellular Adhesion Molecule-1 involved in the adherence of leukocytes to endothelial cells.
  • E-selectin as used herein describes the Endothelial Leukocyte Adhesion Molecule-1 involved in the adherence of leukocytes to endothelial cells.
  • VCAM-1 as used herein describes the Vascular Cell Adhesion
  • Molecule - I involved in adherence of leukocytes (monocytes and neutrophils especially) .
  • mRNA refers to mature, processed mRNA (messenger ribonucleic acid) or unprocessed, nuclear pre-mRNA transcribed from the gene(s) encoding for the synthesis of specific protein, such as ICAM-1, E-selectin, or VCAM-I. These sequences of ribonucleic acid are used to select the antisense oligonucleotide sequences which are complementary to discrete portions of the mRNA or pre-mRNA.
  • mRNA is used herein to indicate either the mature of processed mRNA; or the unprocessed nuclear pre-mRNA.
  • oligonucleotide includes both oligomer of ribonucleotides i.e., oligoribonucleotides, and oligomer of deoxyribonucleotides i.e., oligodeoxyribonucleotides, or oligodeoxynucleotides . Unless otherwise indicated, the term “oligonucleotide” also includes oligomers which may be large enough to be termed "polynucleotides”.
  • oligonucleotide includes oligomer and polymers of biologically significant nucleotides, adenine, deoxyadenine, guanine, deoxyguanine, thymine, uracil, cytosine and deoxycytosine, as well as oligomers and polymers which contain other novel nucleotides and are hybridizable to the target mRNA transcript. These terms also include oligomers and polymers having one or more purine or pyrimidine moieties, sugar moieties, or internucleotide linkage (s) which has or have been chemically modified.
  • This term may be used to include olgionucleotides that are comprised of modifications to the phosphodiester backbone including, but not limited to, phosphorothioate, methylphosphonate, or 2 ',5' linkages of normal phosphodiester or modified phosphodiester nature. Such modifications may be substantial and may encompass nonnucleotide chemistries including non-sugar, non-phosphate backbone, and chemical alterations to the bases to maintain the specific hybridization to the mRNA by base-pairing mechanisms, similar to or different from Watson-Crick base pairing. These terms further include those oligomers and polymers that are composed of nucleoside containing bases joined to the sugar moieties in the alpha configuration.
  • downstream is used herein to indicate the 5' to 3' direction in a nucleotide sequence.
  • upstream indicates the 3' to 5' direction.
  • complementary is used herein to indicate that the oligonucleotide is capable of hybridizing to and forming a stable heteroduplex with its complementary sequence in the mRNA transcript with a Tm of at least 35°C under in vitro conditions of physiological ionic strength.
  • oligonucleotides having base sequences capable of hybridizing to the mRNA transcripts of the human ICAM-1, E-selectin, or VCAM-I adhesion receptors are provided. Hybridization of the oligonucleotide to the mRNA substantially blocks the translation of the mRNA transcript. Because ICAM-1, E-selectin, and VCAM-I are essential for the initial attachment or adhesion of leukocytes arising from stimulations that induce inflammation, down-regulation of the expression of one, both, or all three of these adhesion molecules may be mediated through antisense technologies.
  • the oligonucleotides of the present invention are constructed and purified by methods known in the art.
  • the oligonucleotides may be prepared by solid phase or solution phase synthesis in an automated DNA synthesizer or by solution phase techniques .
  • Example 1 Antisense oligonucleotides were synthesized using standard published techniques for the synthesis of phosphorothioate (PS) oligonucleotides. This oligonucleotide chemistry was employed to identify active sequence locations on the mRNA molecules for ICAM-1, E-selectin, or VCAM-1 and not intended to be the sole modification employable under the scope of this invention. Preferably, synthesis of antisense oligonucleotides is performed using a solid support and a commercially available DNA synthesizer. Antisense oligonucleotides were synthesized using standard phosphoramidite chemistry.
  • PS phosphorothioate
  • the oxidation may be mediated via iodine, while for the synthesis of these phosphorothioates, the oxidation was mediated with a 0.2 M solution of 3H-1, 2-benzodithiole-3-one, 1-dioxide in acetonitrile (Iyer, R.P., et al. (1990) J. Amer. Chem. Soc. 112: 1253-1254) for the step-wise thioation of the phosphate linkages.
  • the thioation step is increased to 68 sec and is followed by a capping step.
  • the trityl-on oligonucleotide is purified using HPLC.
  • HPLC methodologies consisted of chromatography using a PRP-1 column and gradient of acetonitrile in 50 mM triethylammonium acetate, pH 7.0 (4-32% in 30min, flow rate of 1.5 ml/min) . Appropriate fractions were pooled, evaporated, and treated with 5% acetic acid for 15 min at ambient temperature.
  • the oligonucleotide solution was extracted with an equal volume of ethyl acetate, neutralized with ammonium hydroxide, frozen and lyophilized. Solution based chemistries are also useful for synthesis of antisense oligonucleotides and are useful of scaled-up synthesis of oligonucleotides.
  • the oligonucleotide may be prepared through the use of reverse transcriptase, PCR synthesis or via other genetic engineering techniques.
  • the method of preference is an automated DNA synthesis on a solid phase support, however, any method of synthesizing oligonucleotides may be used.
  • the specific oligonucleotide sequences are such that they are complementary to either the ICAM-1, E-selectin, or VCAM-I mRNAs or genes.
  • the oligonucleotide sequences were complementary to the transcripts including the translation initiation codons, and sequences 5' and/or 3' to the translation initiation sites,, of the 5' cap region of the mRNAs and sequences 3' to the cap sites.
  • oligonucleotide sequences were also made complementary to the 3' untranslated region of the genes. Although not tested, it is anticipated by this invention that modifications to antisense oligonucleotides could include cross-linking DNA or intercalating DNA moieties. Furthermore, the invention contemplates that other oligonucleotides or combination of oligonucleotides capable of specifically and substantially inhibiting the expression of ICAM-1, E-selectin, or VCAM-I can be used.
  • the oligonucleotides of this invention comprise predetermined sequences of DNA ranging in size from about 10 bases up to about 30 bases, which is sufficient to define a unique sequence the human target mRNA transcripts.
  • two or more oligonucleotides of 7-15 bases may be joined together by non-nucleotide linkages.
  • less than 14 bases may be used, however the degree of sequence uniqueness decreases rapidly with decreasing length and thereby greatly reduce the specificity of the oligonucleotide for the target mRNA transcript.
  • oligonucleotide sequences greater than about 30 bases may be subject to decreased cellular uptake and have an increased likelihood of containing short stretches of nucleotide sequence capable of forming quasi-stable hybrids with non-target mRNA sequences, other than one of the targeted mRNA transcripts. It is preferable that the oligonucleotides comprise about 14 to 22 bases and most preferably, a 21-mer oligonucleotide is used.
  • HUVEC were cultured to confluence in 48 well plates of 8 well chamber slides using media described by The American Types Culture Collection (ATCC, Rockville, MD) . After washing the cells four times with 200 ⁇ 1 Opti-MEM (Life Technologies, Frederick, MD) , HUVEC in duplicate or triplicate wells were treated for 2 hours at 37°C with Lipofectin (Life Technologies, Frederick, MD) at 10 ⁇ g/ml in Opti-MEM, immediately followed by addition of concentrated antisense phosphorothioate oligodeoxynucleotides (PS-ODN) in phosphate buffered saline with Ca 2+ (PBS) .
  • PS-ODN concentrated antisense phosphorothioate oligodeoxynucleotides
  • This assay has an advantage over assays that measure the activity of the protein since it is a direct quantitation of the presence of the target protein.
  • reductions in the amount of ICAM-1 , E-selectin, or VCAM-1 suggests that translation of the targeted protein's mRNA has been inhibited ' . Since the mechanism of antisense action is to interfere with mRNA translation, this is a better indicator of antisense activity.
  • Example 5 These experiments utilized both Balb/c and C57BL/6 mice. Both types of mice exhibited a reproducible inflammatory response following an i.p. challenge with 2 ml of 1.5% thioglycollate (TG) .
  • TG thioglycollate
  • mice were treated by injection into the tail vein 30 minutes prior to TG challenge with 200 ⁇ 1 of PBS containing 50 ⁇ g of antibody.
  • antisense oligonucleotides 100 ⁇ g oligonucleotide was administered in 200 ⁇ 1 PBS through the tail vein 3-4 hours prior to TG challenge. This dose is approximately 5 mg/kg.
  • Antisense treatment did not involve the use of any liposomal formulations or other delivery vehicles, such as Lipofectin (Life Technologies, Inc.).
  • Lipofectin Life Technologies, Inc.
  • compounds were administered as for single treatment mice.
  • Four hours after TG administration mice were sacrificed by cervical dislocation and the peritoneum was lavaged twice with 3 ml of PBS containing BSA, heparin, and EDTA. The lavages were combined and white blood cells were counted in an automated cell counter. Aliquots were centrifuged in a Cytospin, stained with H&E, and differentials obtained by microscopic examination. The percentages of PMN, lymphocytes, and macrophages were multiplied by total white blood cell counts to calculate the number of individual cell types.
  • Figure 1 illustrates inhibition of monocytes (A, B, C, and D) adhesion to TNFa-stimulated HUVEC by .1 ⁇ M PS-ODNs GM1508 (C) ,
  • PS-ODN were tested for their ability to inhibit the expression of ICAM-1 in HUVEC.
  • oligonucleotides ten (10) different sequences exhibited significant activity (greater than approximately 40% inhibition) at 0.1 ⁇ M oligonucleotide concentration. Many of the tested sequences failed to convincingly produce a reduction in ICAM-1 induction. Based upon the assay above, these oligonucleotides exhibited significant 0 activity in HUVEC for reducing the expression of ICAM-1. Regardless of the inducer chosen (LPS, TNF-a, or ILlb), antisense effects were consistent for active oligonucleotides.
  • inducer chosen LPS, TNF-a, or ILlb
  • E-selectin expression was inhibited by active sequences. This is suggestive of a true antisense mechanism. Also, generally non-antisense effects of PS-ODN are observed at concentrations much greater than 1.0 ⁇ M. In order to identify an active sequence, we chose to use data derived from a single dosing of 0.1 * ⁇ M concentration. In Table 2 the percentage expression for E-selectin is listed for the above oligonucleotides when administered to HUVEC at 0.1 ⁇ M concentrations .
  • Antisense oligonucleotides have activity in reducing the expression of E-selectin when HUVEC are stimulated with various inducers of inflammation. Some oligonucleotides are more active than others. In the sequences identified as being active against E-selectin expression the most potent two were Sequence ID No. 16 and Sequence ID No. 20. Many PS-ODN were tested for their ability to inhibit the expression of VCAM-1 in HUVEC. Within this group of oligonucleotides, twenty (20) different sequences exhibited significant activity (about 40% inhibition) in the 0.1 ⁇ M range. These are listed in Table 3.
  • lavage fluids Prior to challenge with thioglycollate, lavage fluids only contained 90,000 PMN (in addition to 2.5 xlO 6 lymphocytes and 1.2 x 10 6 macrophages) . After TG challenge, the numbers of PMN rise over four hours to a peak of 9-11 x 10 6 , and then slowly recede over the next 48-72 hours. The numbers of lymphocytes and macrophages increase to a much lesser extent. The infiltration of PMN responds to TG in a dose dependent fashion reaching a maximal response at 1.5 ml of 2% TG. Interperitoneal administration of antibody or antisense oligonucleotides was not found to be effective, so i.v. administration through the tail vein was utilized.
  • oligonucleotide sequences were synthesized targeting various regions of the mRNA. These sequences may not be the best selection for eliciting an antisense response. However, using control sequences containing scrambled or base-pair mismatches sequences, the oligonucleotide-induce effect is much less (DPC5205, DPC 5206, and DPC5094, Table 5).
  • oligonucleotide DPC5028 Treatment with antisense oligonucleotide DPC5028, a 21-mer directed at the AUG initiation codon of murine ICAM-1, was successful at inhibiting the adherence of PMN following TG ' challenge (Table 5).
  • the effect of DPC5028 was dependent -upon the time of administration.
  • Oligonucleotide DPC5028 was effective when administered between 24 hours before and up to the time of TG challenge. At 48 hours prior to challenge and 2 hours post challenge, DPC5028 was without effect. However, due to varying sensitivities of the experimental subjects, there was intra-experimental variability. During these experiments, it was noticed that there was an age dependent incidence of skin inflammation in C57BL/6 mice that was later identified by Jackson Laboratories.
  • mice Due to this complication, data generated from these animals was suspect. Also, the Balb/c mice exhibited an age dependent anergy, in that as these mice age there is a reduction in their response to TG challenge. Over a 30 day period the number of PMN was found to decrease by up to 25% following TG challenge. During this time period, there was an attenuation of the anti-ICAM-1 blockade evidenced by anti-ICAM-1 antibody or DPC5028 pre-treatment. In experiments conducted on younger Balb/c mice there was a significant (compared to TG treated mice) reduction in PMN adherence with DPC5028 treatment (Table 5) .
  • phosphorothioate oligonucleotides can elicit sequence-specific effects through non-antisense mechanisms when the sequence contains either a CpG motif (for example see Kreig et al., 1995, Nature 374: 546) .
  • the CpG motif can cause immune activation, particularly activation of B-cells and natural killer cells.
  • DPC5028 does not contain any CpG motif so this sequence should not be inducing activation of the immune response.
  • two different sequences containing one (DPC5207) or three (DPC5083) CpG motifs were tested for their effects on PMN adhesion in this model system, there was a significant reduction in PMN adhesion (Table 5) . Since DPC5028 and the other control oligos do not contain this motif, the activity of DPC5028 may be a result of antisense-mediated reductions in ICAM-1 expression.

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Abstract

La présente invention concerne un traitement contre des maladies et des pathologies associées chez l'homme à des inflammations, à savoir, notamment l'acné, le psoriasis, l'arthrite, le rejet de greffe, les blessures, les brûlures, le choc septique et les complications inflammatoires du choc. L'invention concerne également un procédé permettant d'inhiber sélectivement l'expression des produits de transcription d'ARNm de la molécule d'adhésion intercellulaire humaine ICAM-1, de la sélectine E humaine ou de la molécule d'adhésion cellulaire vasculaire humaine VCAM-1 au moyen d'au moins un oligonucléotide complémentaire d'au moins une partie des ARNm de l'ICAM-1 humaine, de la sélectine E humaine ou de la VCAM-1 humaine. L'invention concerne en outre, non seulement des oligonucléotides complémentaires de certaines parties des ARNm de l'ICAM-1 humaine, de la sélectine E humaine ou de la VCAM-1 humaine, mais aussi des compositions comprenant ces oligonucléotides.
EP96945380A 1996-12-02 1996-12-02 Inhibition antisens de molecules d'adhesion humaines Withdrawn EP0950060A4 (fr)

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/US1996/019194 WO1998024797A1 (fr) 1996-12-02 1996-12-02 Inhibition antisens de molecules d'adhesion humaines

Publications (2)

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EP0950060A1 true EP0950060A1 (fr) 1999-10-20
EP0950060A4 EP0950060A4 (fr) 2000-07-05

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EP (1) EP0950060A4 (fr)
JP (1) JP2001505432A (fr)
AU (1) AU1564897A (fr)
CA (1) CA2273203A1 (fr)
WO (1) WO1998024797A1 (fr)

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ATE236250T1 (de) * 1998-09-25 2003-04-15 Deutsches Krebsforsch Antisense-sequenzen für die hemmung der expression des adhäsionsmoleküls icam-1
DE19844111A1 (de) * 1998-09-25 2000-04-20 Deutsches Krebsforsch Antisense-Sequenzen für die Hemmung der Expression des Adhäsionsmoleküls ICAM-1
US6114517A (en) * 1998-12-10 2000-09-05 Isis Pharmaceuticals Inc. Methods of modulating tumor necrosis factor α-induced expression of cell adhesion molecules
US6670321B1 (en) 1998-12-30 2003-12-30 The Children's Medical Center Corporation Prevention and treatment for retinal ischemia and edema
WO2000044409A1 (fr) * 1999-01-27 2000-08-03 University College London Formulations comportant des nucleotides anti-sens de connexine
DE10019252A1 (de) * 2000-04-18 2001-10-31 Klaus Karl Degitz Polydesoxyribonukleotide zur Hemmung der ICAM-1-Genexpression
DE10141443B4 (de) * 2001-08-23 2007-02-01 Christos C. Prof. Dr. Zouboulis Verwendung von molekularbiologisch hergestellten, nicht-viralen Wirkstoffen zur Behandlung der Akne
EP2314689A3 (fr) 2003-12-03 2012-12-19 Coda Therapeutics (NZ) Ltd Composés anti-sens ciblés contre les connexines et procédés d'utilisation associés
US9248141B2 (en) 2005-02-03 2016-02-02 Coda Therapeutics, Inc. Methods of treatment by administering anti-connexin proteins and mimetics
KR20150072458A (ko) 2006-11-15 2015-06-29 코다 테라퓨틱스, 인크. 상처 치유를 위한 개선 방법 및 조성물
KR101521306B1 (ko) 2006-12-11 2015-05-18 코다 테라퓨틱스, 인크. 치유 부전 상처를 치유하는 조성물로서의 항-커넥신 폴리뉴클레오티드
JP2011507857A (ja) 2007-12-21 2011-03-10 コーダ セラピューティクス， インコーポレイテッド 線維症性の状態の処置のためのコネキシン４３の阻害剤の使用
US10465188B2 (en) 2014-08-22 2019-11-05 Auckland Uniservices Limited Channel modulators
CA3061738A1 (fr) 2017-04-28 2018-11-01 Auckland Uniservices Limited Methodes de traitement et nouvelles constructions

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JP2001505432A (ja) 2001-04-24
AU1564897A (en) 1998-06-29
CA2273203A1 (fr) 1998-06-11
WO1998024797A1 (fr) 1998-06-11
EP0950060A4 (fr) 2000-07-05

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