WO1996015242A2 - Procedes d'inhibition de la proliferation cellulaire par inhibition de l'activite mitogene du facteur inhibiteur de la migration des macrophages - Google Patents

Procedes d'inhibition de la proliferation cellulaire par inhibition de l'activite mitogene du facteur inhibiteur de la migration des macrophages Download PDF

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WO1996015242A2
WO1996015242A2 PCT/US1995/015000 US9515000W WO9615242A2 WO 1996015242 A2 WO1996015242 A2 WO 1996015242A2 US 9515000 W US9515000 W US 9515000W WO 9615242 A2 WO9615242 A2 WO 9615242A2
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mif
nucleic acid
inhibitory nucleic
cell
mrna
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PCT/US1995/015000
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WO1996015242A3 (fr
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Graeme J. Wistow
Vishwas Paralkar
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The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services
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Priority to AU43657/96A priority Critical patent/AU4365796A/en
Publication of WO1996015242A2 publication Critical patent/WO1996015242A2/fr
Publication of WO1996015242A3 publication Critical patent/WO1996015242A3/fr

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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
    • C12N15/1136—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 growth factors, growth regulators, cytokines, lymphokines or hormones
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates in general to the fields of cellular and molecular biology and, in particular, to methods of inhibiting the growth of cells by inhibiting the mitogenic activity of macrophage migration inhibitory factor.
  • oncogenes such as src
  • src encode a protein kinase activity that resembles the protein kinase activity induced by certain growth factors.
  • some peptide growth factors such as PDGF, induce the expression of oncogenes; and certain malignant cells require less stimulation by growth factors than normal cells to proliferate.
  • Peptide growth factors act by binding to receptors on the cell surface. From there, the induction of mitogenesis involves a complex and only partially understood sequence of intra-cellular signals and events. Growth factors induce the expression of many genes, some of which are activated within minutes, called primary response genes, and others * which are activated after a few hours, called delayed early response genes. (See, e.g., Lanahan et al., (1992) Molec . and Cell Biol . , 12:3919-3929.) The function of many of the proteins encoded by these genes is unknown.
  • MIF macrophage migration inhibitory factor
  • MIF macrophage migration inhibitory factor
  • This invention provides methods useful for inhibiting the growth of a cell involving the step of inhibiting the mitogenic activity of MIF in the cell.
  • the methods can be used both in vitro to control growth of transformed cells or cells stimulated growth factors, or the method can be used in vivo in therapeutic methods.
  • the step of inhibiting the mitogenic activity of MIF involves providing the cell with an inhibitory nucleic acid that inhibits expression of MIF.
  • the inhibitory nucleic acid can be a sense or an antisense nucleic acid that inhibits transcription of the MIF gene or translation of MIF mRNA.
  • Antisense nucleic acids can be directed, for example, against a target sequence in the sense strand of the MIF gene, a target sequence spanning the boundary between an intron and an exon of MIF pre-m NA, or, in a preferred embodiment, a target sequence in MIF mRNA.
  • the antisense nucleic acid also can be a ribozyme that cleaves MIF mRNA.
  • This invention also provides isolated forms of these inhibitory nucleic acids. This invention contemplates delivering inhibitory nucleic acids to the cell by introducing an expression vector having an expression control sequence operatively linked to a nucleic acid sequence encoding the inhibitory nucleic acid. It also contemplates contacting the cell with the inhibitory nucleic acid, for example, by administration to the external milieu of the cell.
  • the step of inhibiting MIF activity involves inhibiting the binding of MIF to retinoblastoma CR-h" protein.
  • This invention is also directed to therapeutic methods useful for inhibiting the growth of a malignant cell in an individual involving administering to the individual a therapeutically effective amount of an agent that inhibits the mitogenic activity of MIF in the malignant cell.
  • the agent can be an inhibitory nucleic acid or a compound that interferes with MIF binding to Rb protein.
  • the agent is injected into a tumor mass.
  • This invention also provides pharmaceutical compositions having an agent that inhibits the mitogenic activity of MIF and a pharmaceutically acceptable carrier.
  • Fig. 1 presents results of PAGE on cell extracts from NIH 3T3 cells grown in the presence and absence of PDGF and TGF/31. Treatment of serum-free NIH 3T3 cells with either PDGF or TGF31 results in stimulation of MIF expression.
  • Fig. 2 shows stimulation of the human MIF gene promoter by PDGF and TGFjSl.
  • NIH 3T3 cells were stably transfected with plasmid containing the human MIF gene promoter (V. Paralkar and G. Wistow, (1994) Genomics 19:48-51) cloned upstream of the bacterial reporter gene chloramphenicol acetyltransferase (CAT) .
  • CAT chloramphenicol acetyltransferase
  • Treatment of these cells resulted in four-fold induction of CAT activity over serum-free control in the presence of serum and 2-3 fold in the presence of TGF/31 and PDGF, respectively. Similar results were seen in transient transfection experiments.
  • CAT activity relative to serum-free control is shown as mean values + standard deviation for three individual measurements.
  • Figs. 3A-3B show the effect of treatment with antisense MIF oligonucleotide in abolishing cell proliferation and synthesis of MIF protein.
  • Fig. 3A shows 3 H-thymidine incorporation in NIH 3T3 cells treated with growth factors and antisense or control oligonucleotides.
  • Cells were grown in the presence of 0.2% serum and then exposed to (a) no additional treatment, (b) PDGF (5 ng/ml), (c) PDGF + aMIFl (10 ⁇ M) , (d) PDGF + sMIFl (10 ⁇ M) , (e) PDGF + aMIF2 (10 ⁇ M) , (f) PDGF + lFIMa, (g) TGF/31 (10 ng/ml) and (h) TGF/31 + aMIFl.
  • Antisense treated cells recovered, i.e., they regained the ability to grow after washing with fresh medium: (i) control cells were grown for 48 hr in 10% serum, (j) cells were first treated with PDGF + aMIFl for 24 hours as in part (c) then medium was replaced with 10% serum for a further 24 hours. 3 H-thymidine incorporation is shown relative to value for lane (a) . Each point represents mean ⁇ standard deviation for three measurements. The assay was repeated with multiple preparations of oligos synthesized on two different Applied Biosystems synthesizers giving similar results.
  • Fig. 3B shows a western blot of MIF protein in
  • NIH 3T3 cells grown in the presence of PDGF and TGF ⁇ l and presence or absence of aMIFl using antisera to human MIF.
  • Serum-free NIH 3T3 cells were cultured in the presence of PDGF (10 ng/ml) or TGF01 (15 ng/ml) and presence or absence of aMIFl (10 ⁇ M) overnight.
  • Cells were lysed at 4°C in a buffer containing 1% Nonidet P-40, 50 mM Tris-HCl, pH 7.6, 2 mM EDTA, 1 mM PMSF, 20 ⁇ g/ml aproteinin, 20 ⁇ g/ml leupeptin. Cell lysates were clarified by centrifugation and their protein concentration determined.
  • Protein extracts (10 ⁇ g/treatment) were subjected to SDS PAGE (Novex, San Diego, CA) , transferred to nitrocellulose filters and analyzed by western blotting with chicken anti-human MIF antiserum. This antiserum was raised as an IgY fraction in chickens immunized with a fusion protein of human MIF and maltose binding protein. Visualization was by enhanced chemiluminescence (Amersham, Arlington Heights, IL) , using goat anti-chicken immunoglobulin secondary antibody (Vector labs) .
  • Figs. 4A-4B demonstrate that MIF acts near the Gl/S boundary and interacts with Rb protein.
  • Fig. 4A shows a western blot of cyclins in NIH 3T3 cells grown in the presence of PDGF and presence or absence of aMIFl using anti-cyclin antibodies.
  • Non-specific bands are Ig bands from immunoprecipitation.
  • Fig. 4B shows a flow cytometry analysis of aMIFl treated cells: a) control NIH 3T3 cells in the absence of serum; b) serum-free cells grown in the presence of 5 ng/ml PDGF; c) PDGF-treated cells in the presence of aMIFl. Percentages of cells in GI, S and G2/M phases of the cell cycle are indicated. Cells were grown as described herein. Flow cytometry was performed as described in V.V. Ogryzko et al. (1994) 3. Virol . 68:3724, using a FACscan flow cytometer equipped with a doublet discrimination (Becton-Dickinson) . Fig.
  • Fig. 6A shows a western blot of MIF in immunoprecipitates of NIH 3T3 cell lysates using antisera to cell cycle components which control Gl/S transition. Rabbit immunoglobulin-derived bands from the immunoprecipitation were detected in all lanes. Lane C shows results for western blot of anti-Rb antibody in the absence of cell lysate as control.
  • Lane 1 shows western blot of proteins immunoprecipitated with agarose conjugated antiserum to Rb, retinoblastoma protein
  • Lane 2 shows western blot of protein immunoprecipitated with anti-cyclin E in the presence of agarose conjugated protein A
  • Lane 3 shows western blot of proteins immunoprecipitated with agarose conjugated anti-CDK2. All lanes were subjected to western analysis with chicken anti-human MIF.
  • Fig. 6B shows a western blot of MIF in proteins immunoprecipitated by agarose conjugated anti-Rb under non- reducing conditions.
  • lane C is control without cell lysate while lane 1 contains anti-Rb immunoprecipitate.
  • the immunoglobulin-derived bands now migrate at larger size appropriate for intact antibodies.
  • MIF reactive protein in the anti-Rb immunoprecipitate migrates at a size appropriate for a homodimer under non-reducing conditions.
  • This invention is directed to new methods of inhibiting itogenesis in a cell.
  • the invention results from the discovery that MIF has hitherto unrecognized mitogenic functions and that the inhibition of MIF activity in a cell results in the inhibition of cell proliferation.
  • mitogenesis normally induced by stimulation with growth factors, such as Platelet-Derived Growth Factors (PDGF) and Transforming Growth Factors- ⁇ (TGF- ⁇ )
  • PDGF Platelet-Derived Growth Factors
  • TGF- ⁇ Transforming Growth Factors- ⁇
  • mitogenesis in cells transformed with the oncogenes ras , raf and sis are inhibited when MIF activity is inhibited with antisense polynucleotides.
  • PDGF and TGF- ⁇ l act through different signaling pathways: tyrosine kinase receptors and serine/threonine kinase receptors, respectively.
  • Oncogenes ras , raf and sis act as components early in the signalling pathways that lead to cell division. The fact that inhibition of MIF halts cell division in all these pathways indicates that MIF acts late in the cell cycle after these pathways converge.
  • results described herein show that MIF binds to the retinoblastoma ("Rb") protein in the cell and is required for entry of the cell into the S phase of the cell cycle.
  • Rb retinoblastoma
  • Inhibiting MIF activity arrests cells in GI phase, and they cease proliferation.
  • this invention provides methods that result in the inhibition of cell growth involving inhibiting the mitogenic activity of MIF in a cell, both in vitro and in vivo .
  • the inhibition of MIF activity of cells in vitro is useful as a research tool to investigate the complex sequence of events that regulate the cell cycle.
  • the inhibition of MIF activity in cells in vivo is useful in therapeutic methods for inhibiting the growth of malignant cells.
  • the methods of this invention are applicable to any culture cells whose growth has been stimulated by growth factors, such as, for example, PDGF,
  • TGF-j ⁇ Fibroblast Growth Factor
  • FGF Fibroblast Growth Factor
  • IGF Insulin-like Growth Factors
  • FGF Fibroblast Growth Factor
  • IGF Insulin-like Growth Factors
  • the methods of this invention are applicable, for example, to malignant cells.
  • this invention contemplates inhibiting the growth of cells whose malignancy involves uncontrolled progression through S phase, including those cells expressing oncogenes such as c- ras , c-raf and c-sis .
  • the methods of this invention are also applicable to inhibiting the growth of cells in vivo whose growth is induced by growth factors.
  • MIF or “macrophage migration inhibitory factor” means the human protein having lymphokine activity and having the DNA sequence [SEQ ID NO:l] and deduced amino acid sequence [SEQ ID NO:2] presented in Table I, its alleles and any mammalian cognates of it.
  • the sequence in Table I is from V. Paralkar and G. Wistow, (1994) Genomics , 19:48-51 (GenBank Accession No. L19696) .
  • "#” indicates the transcription start site.
  • “*” indicates the beginning and end of introns.
  • " indicates the polyadenylation site (AATAAA) .
  • the sequence of other MIF cDNAs are also known.
  • R L R 1 S P D R* 1621 GCCTGCGCAT CAGCCCGGAC AGGTACGCGG AGTCGCGGAG GGGCGGGGGA GGGGCGGCGG
  • This invention contemplates inhibiting MIF activity in a cell by, for example, inhibiting MIF expression with inhibitory nucleic acids or by inhibiting the binding of MIF to Rb protein.
  • methods for inhibiting the mitogenic activity of MIF in a cell involve providing the cell with an inhibitory nucleic acid that inhibits expression of MIF.
  • Inhibitory nucleic acids are single-stranded nucleic acids which can specifically hybridize to a target nucleic acid sequence. Typically, hybridization will occur when there is at least about 55% complementarity 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, (1984) Nucleic Acids Res . 12:203.
  • RNA-RNA, DNA-DNA, or RNA-DNA duplex By binding to the appropriate target sequence, an RNA-RNA, DNA-DNA, or RNA-DNA duplex is formed.
  • RNA-RNA, DNA-DNA, or RNA-DNA duplex is formed.
  • antisense because they are usually complementary to the sense or coding strand of the gene.
  • inhibitory nucleic acids refers to both “sense” and “antisense” nucleic acids.
  • the inhibitory nucleic acid can inhibit the function of the target nucleic acid. This could, for example, be a result of blocking DNA replication or transcription; interfering with processing of, poly(A) addition to, or translation of mRNA; or promoting inhibitory mechanisms of the cells, such as promoting RNA degradation.
  • Inhibitory nucleic acid methods therefore encompass a number of different approaches to altering expression of specific genes that operate by different mechanisms. These different types of inhibitory nucleic acid technology are described in C. Helene and J. Toulme, (1990) Biochim . Biophys . Acta . , 1049:99-125, which is referred to hereinafter as "Helene and Toulme".
  • Inhibitory nucleic acid approaches can be classified into those that target DNA sequences, those that target RNA sequences (including pre-mRNA and mRNA) , those that target proteins (sense strand approaches) , and those that cause cleavage or chemical modification of the target nucleic acids.
  • Approaches targeting DNA fall into, several categories.
  • Nucleic acids can be designed to bind to the major groove of the duplex DNA to form a triple helical or "triplex" structure.
  • inhibitory nucleic acids are designed to bind to regions of single stranded DNA resulting from the opening of the duplex DNA during replication or transcription. See Helene and Toulme.
  • this invention is directed to methods in which the inhibitory nucleic acid is directed against a target DNA sequence in the sense strand of the MIF gene.
  • the target DNA sequence can span the site of initiation of transcription, nucleotide number 1077 of SEQ ID NO:l.
  • inhibitory nucleic acids are designed to bind to mRNA or mRNA precursors. Inhibitory nucleic acids are used to prevent maturation of pre-mRNA. Inhibitory nucleic acids can be designed to interfere with RNA processing, splicing or translation.
  • inhibitory nucleic acids complementary to sequences at the boundary between introns and exons of the gene can be used to interfere with cleavage of pre-mRNA and ligation of exons.
  • this invention is directed to methods in which the inhibitory nucleic acid is directed against a sequence spanning the boundary between an intron and an exon of MIF pre-mRNA. Table I indicates the location of introns and exons in the MIF gene.
  • the inhibitory nucleic acids can also be targeted against mRNA.
  • the inhibitory nucleic acids are designed to hybridize to a target sequence in MIF mRNA and, thereby, interfere with its translation. Translation of mRNA is inhibited if there is a measurable decrease in the amount of MIF protein produced by the cell after treatment. Inhibiting translation of an mRNA results in suppression of the cellular function carried out by the encoded protein.
  • an inhibitory nucleic acid complementary to regions of c-myc mRNA inhibits c-myc protein expression in a human promyelocytic leukemia cell line, HL60, which over-expresses the c-myc proto-oncogene. See E.L.
  • this invention is directed to methods in which the inhibitory nucleic acid is directed against a target sequence in MIF mRNA.
  • Inhibitory nucleic acids for inhibiting the translation of mRNA can have at least about 15 nucleotides. They also can be as long as the entire target mRNA. In one embodiment of the invention, the inhibitory nucleic acid is about 30 nucleotides long.
  • an inhibitory nucleic acid depends, in part, on the location of the target sequence in the mRNA.
  • Inhibitory nucleic acids that hybridize to MIF mRNA at the translation start site, i.e., that are directed against a sequence that includes the AUG start codon, are particularly effective in inhibiting translation of the MIF mRNA.
  • this invention provides the antisense inhibitory nucleic acid having the sequence GATGAACATA GGCATGGTGG
  • CGGAGAGACT [SEQ ID NO:3].
  • the underlined sequence, CAT hybridizes to the target ATG start codon and, thereby, the polynucleotide spans the start codon.
  • Other antisense inhibitory nucleic acids directed against the start codon include fragments of the aforementioned antisense nucleic acid of at least about 15 nucleotides, as well as other nucleic acids having a sequence that hybridizes to a sequence of about 15 nucleotides that span the start codon in the MIF mRNA sequence.
  • the verb "to have,” when describing the sequence of a nucleic acid molecule, is used in the narrow sense to mean that the nucleic acid molecule has the sequence of nucleotides given without flanking sequences. Inhibitory nucleic acids directed against other parts of the MIF mRNA, particularly the 3' untranslated region, are less effective and, therefore, are to be avoided.
  • the inhibitory nucleic acids introduced into the cell can also encompass fragments of the "sense" strand of the gene or mRNA to trap or compete for the enzymes or binding proteins involved in mRNA translation. Of course, these fragments do not encode functional proteins or the target function would not be inhibited. See Helene and Toulme. Accordingly, this invention is directed to methods in which the inhibitory nucleic acid includes sequences from the sense strand of the MIF gene or MIF mRNA. Also, this invention provides methods in which the inhibitory nucleic acids induces chemical inactivation or cleavage of the target genes or mRNA. Chemical inactivation can occur by the induction of crosslinks between the inhibitory nucleic acid and the target nucleic acid within the cell.
  • irreversible photochemical reactions can be induced in the target nucleic acid by means of a photoactive group attached to the inhibitory nucleic acid.
  • Other chemical modifications of the target nucleic acids induced by appropriately derivatized inhibitory nucleic acids can also be used.
  • the inhibitory nucleic acid can be attached to manganese or ferric ions.
  • this invention is directed to methods wherein the inhibitory nucleic acid is attached to an agent that effects chemical inactivation of MIF mRNA.
  • Cleavage, and therefore inactivation, of the target nucleic acids can be effected by attaching a substituent to the inhibitory nucleic acid which can be activated to induce cleavage reactions.
  • the substituent can be one that effects either chemical, photochemical or enzymatic cleavage.
  • cleavage can be induced by the use of ribozymes or catalytic RNA.
  • the inhibitory nucleic acids would contain either naturally occurring RNA (ribozymes) or synthetic nucleic acids with catalytic activity.
  • This invention also provides methods in which the inhibitory nucleic acid cleaves MIF mRNA. Bratty et al., (1992) Biochim . Biophys . Acta . , 1216:345-59 (1993) and Denhardt, (1992) Ann . N. Y. Acad. Sci . , 660:70-76 describe methods for making ribozymes.
  • inhibitory nucleic acids for use in the methods of this invention by any means known to the art including, for example, chemical synthesis, PCR and expression from expression vectors.
  • Nucleic acid synthesis methods are well known to those of skill in the art.
  • Inhibitory nucleic acids are chemically synthesized according to the solid phase phosphoramidite triester method first described by S.L. Beaucage and M.H. Caruthers, (1981) Tetrahedron Letts . , 22(20) :1859-1862 using an automated synthesizer, as described in D.R. Needham-VanDevanter et al . , (1984) Nucleic Acids -Res., 12:6159-6168.
  • nucleic acids Purification of nucleic acids is by either native acrylamide gel electrophoresis or by anion-exchange HPLC as described in J.D. Pearson and F.E. Regnier, (1983) J . Chrom . , 255:137-149.
  • Inhibitory nucleic acids can also be produced by using polymerase chain reaction (PCR) technology. Appropriate primers and probes for amplifying the nucleic acid region of choice are generated from analysis of the DNA sequences. Nucleic acid primers complementary to the two 3' borders of the DNA region to be amplified are synthesized. The polymerase chain reaction is then carried out using the two primers. See PCR Protocols : A Guide to Methods and Applications (Innis, M, Gelfand, D.
  • PCR amplified DNA can then be denatured and the antisense strand isolated by standard techniques known to those of skill in the art. See Sambrook, et al . , supra .
  • sequence of the inhibitory nucleic acids can be verified using the chemical degradation method of Maxa and Gilbert, 1980, in Grossman, L. and Moldave, D. , eds. Academic Press, New York, Methods in Enzymology, 65:499-560.
  • the inhibitory nucleic acids can be conventional nucleic acids, or are more commonly nucleic acids having properties which make them more desirable for inhibitory nucleic acid activity. For example, they can be made resistant to nucleases or more capable of specific binding to the desired target sequences. The specific binding can be effected by providing inhibitory nucleic acids having sequences which result in conventional base-pairing, or which recognize double-stranded DNA by binding to the major or minor grooves which are present in the DNA double helix. Alternatively, the inhibitory nucleic acids, in either single stranded or duplex form, can recognize target protein.
  • oligo ers with physiological properties which render them more effective.
  • the general approach to constructing various nucleic acids useful in inhibitory nucleic acid therapy has been reviewed by A.R. Vander Krol et al . (1988), Biotechnique ⁇ 6:958-976, and by CA. Stein et al . , (1988) Cancer Res . (1988) 48:2659-2668. See also Oligodeoxynucleotides : Antisense Inhibitors of Gene Expression, Cohen, J.S., editor, MacMillan Press, London, pages 79-196 (1989), and Antisense RNA and DNA, (1988), D.A. Melton, Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
  • nucleic acids are synthesized with alternative linkages other than the conventional phosphodiester linkage.
  • methylphosphonates wherein one of the phosphorous-linked oxygens has been replaced by methyl phosphorothioates, wherein sulfur replaces one of the oxygens
  • various amidates wherein NH 2 or organic amino derivatives, such as morpholidates or piperazidates, replace an oxygen.
  • carbonate and carbamate linkages can be employed, as well as those involving sulfur rather than oxygen as a linking substituent.
  • Other modified internucleotide linkages can be synthesized that are generally nuclease resistant. See Miller et al . , (1981) Biochemistry 20:1874-1880, Letsinger et al . ,
  • Nuclease stability can also be obtained through alternate modifications.
  • Haralambidis et al . Haralambidis et al . ,
  • inhibitory nucleic acids resistant to exo- and endo- nucleases, including sulfur substitution of a deoxyribose phosphodiester oxygen (phosphorothioate nucleotides) , and synthesis of the nucleosides in the ( ⁇ )-anomeric configuration rather than the natural (/3)-anomers.
  • phosphorothioate nucleotides phosphorothioate nucleotides
  • synthesis of the nucleosides in the ( ⁇ )-anomeric configuration rather than the natural (/3)-anomers for example, the phosphorothioate modification of inhibitory nucleic acids is used.
  • This form although resistant to most nucleases, does not interfere with RNase H cleavage of endogenous mRNA.
  • RNase H degradation of the endogenous mRNA component of an (antisense) DNA:RNA hybrid can significantly amplify the inhibition of mRNA translation by the inhibitory nucleic acid caused by stearic hinderance.
  • the stability, nuclease resistance, and efficiency of the inhibitory nucleic acids can be improved by coupling an intercalating agent such as 2-methoxy, 6-chloro, 9-aminoacridine to the 3' or 5' end.
  • the acridine conjugate can also promote the passage of the inhibitory nucleic acid out of the endocytic compartment and into the cytoplasm. Therefore, preferably, acridine-conjugated phosphothiorate antisense oligodeoxyribonucleotides with specificity for key activation and effector molecules in the targeted autoreactive T cells are constructed.
  • Intercalators and chelators which enhance the ability of the nucleic acid to bind the target DNA or RNA can also be introduced as a modification. These substituents can be attached to the 5' end of preconstructed nucleic acids using amidite or H-phosphonate chemistry, as described by K.K. Ogilvie et al . (1987) Pure and Appl . Chem . , 59:325, and by B.C. Froehler (1986) Nucleic Acids Res . 14:5399. Intercalators can also be attached to the 3' end of oligomers, for example as described by U. Asseline et al. (1989), Tet . Lett . , 30:2521.
  • substituents can be bound to the 3' end of oligomers by alternate methods.
  • disulfides have been used to attach various groups to the 3' terminus, as described by R. Zuckerman et al . , (1987) Nucleic Acids Res . 15:5305. It is known that nucleic acids which are substituted at the 3 ' end show increased stability and increased resistance to degradation by exonucleases (G. Lancelot et al . (1985) Biochemistry 24:2521; U. Asseline et al . (1984) , Proc . Natl . Acad . Sci . USA, 81:3297) .
  • Nucleic acids also can be synthesized containing a variety of pseudonucleotides or pseudonucleosides which can confer desirable properties such as nuclease resistance.
  • the pseudonucleotides or pseudonucleosides can also be used as a means to conjugate other molecules to the nucleic acid, such as interchelators. See International Patent Application, No. WO 91/13080, Lin, et al . entitled "Pseudonucleosides and Pseudonucleotides and their Polymers”.
  • the methods of this invention contemplate a variety of means for delivering the inhibitory nucleic acid to the cell including, for example, direct uptake of the molecule by the cell from solution, facilitated uptake through liposome vectors and intracellular expression from an expression cassette.
  • Soluble inhibitory nucleic acids present in the external milieu have been shown to gain access to the cytoplasm and inhibit translation of specific mRNA species.
  • this invention provides expression vectors having an expression control sequence operatively linked to a nucleic acid sequence that encodes an inhibitory nucleic acid that inhibits expression of MIF.
  • An expression control sequence is operatively linked to a nucleic acid sequence when it directs the transcription and translation of that sequence in an appropriate host cell.
  • Expression vectors useful in this invention depend on their intended use. Expression vectors must, of course, contain expression and replication signals compatible with the host cell.
  • Expression vectors useful in this invention include viral vectors, such as retroviruses, adenoviruses and adeno-associated viruses; plasmid vectors; cosmids; liposomes and the like. Viral and plasmid vectors are preferred for transfecting mammalian cells.
  • Appropriate expression control sequences for mammalian cells include, for example, the SV40 promoter, the RSV (Rous sarcoma virus) promoter and the CMV (cytomegalovirus) promoter.
  • This invention also provides methods for inhibiting the activity of MIF in a cell by inhibiting the binding of MIF to Rb protein.
  • These compounds can be, for example, peptides or other organic molecules.
  • the compounds can also be antibodies or engineered binding fragments of antibodies, such as Fab fragments. Small molecules can spontaneously enter cells, while larger molecules may require vectors, such as liposomes, to gain entry.
  • Such a test can involve, for example, incubating a sample mixture of the compound to be tested and either MIF or Rb protein, adding Rb protein or MIF, respectively, to the sample mixture, and determining whether the binding of MIF to Rb protein has been inhibited compared to a control sample.
  • Peptides can be identified by screening a peptide library.
  • methods of inhibiting the activity of MIF are useful in therapeutic treatments for inhibiting the growth of malignant cells in an individual. These treatments involve administering to the individual an effective amount of an agent that inhibits the mitogenic activity of MIF in the cells.
  • Agents useful in these methods include, for example, an inhibitory nucleic acid of this invention or a compound that inhibits binding of MIF to Rb protein.
  • this invention contemplates the use of expression vectors, preferably retroviral vectors, for delivery of the inhibitory nucleic acid molecules of this invention.
  • Methods for delivering nucleic acids as part of genetic therapy are known in the art, as described above.
  • One can administer compounds that inhibit the mitogenic activity of MIF in the form of pharmaceutical compositions including the compound and a pharmaceutically acceptable carrier.
  • injection is preferred, including intramuscular, intravenous, intraperitoneal, and subcutaneous injection.
  • Suitable formulations for injection are found in Remington ' s Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, PA, 17th ed. (1985) .
  • a variety of pharmaceutical compositions comprising agents of the present invention and pharmaceutically effective carriers can be prepared.
  • the pharmaceutical compositions are suitable in a variety of drug delivery systems. For a brief review of present methods of drug delivery, See , Langer, (1990) Science 249:1527-1533.
  • the agents of the present invention can be prepared as formulations in pharmaceutically acceptable media, for example, saline, phosphate buffered saline. Hank's solution, Ringer's solution, dextrose/saline, glucose solutions and the like.
  • the compositions can contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as buffering agents, tonicity adjusting agents, wetting agents, detergents and the like.
  • Additives can also include additional active ingredients such as bactericidal agents, or stabilizers.
  • Systemic administration can also be by transmucosal or transdermal means, or the agents can be administered orally.
  • penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrations are generally known in the art, and include, for example, for transmucosal administration, bile salts and fusidic acid derivatives. In addition, detergents can be used to facilitate permeation.
  • Transmucosal administration can be through nasal sprays, for example, or using suppositories.
  • the agents are formulated into conventional oral administration such as tablets, capsules and tonics.
  • compositions of the present invention it can be desirable to modify the complexes of the present invention to alter their pharmacokinetics and biodistribution.
  • pharmacokinetics See, Remington ' s Pharmaceutical Sciences , supra , Chapters 37-39.
  • a number of methods for altering pharmacokinetics and biodistribution are known to one of ordinary skill in the art (See, e .g. , Langer, supra) . Examples of such methods include protection of the complexes in vesicles composed of substances such as proteins, lipids (for example, liposomes) , carbohydrates, or synthetic polymers.
  • inhibitory nucleic acids can be incorporated into liposomes in order to enhance their pharmacokinetics and biodistribution characteristics.
  • Liposome charge is an important determinant in liposome clearance from the blood, with negatively charged liposomes being taken up more rapidly by the reticuloendothelial system (Juliano, (1975) Bioc e-m. Biophys . Res . Commun . 63:651) and thus having shorter half-lives in the bloodstream. Liposomes with prolonged circulation half-lives are typically desirable for therapeutic and diagnostic uses. For instance, liposomes which can be maintained from 8, 12, or up to 24 hours in the bloodstream are particularly preferred. A variety of methods are available for preparing liposomes, as described in, e.g., Szoka et al . , (1980) Ann. Rev . Biophys . Bioeng.
  • the liposomes are prepared with about 5-15 mole percent negatively charged phospholipids, such as phosphatidylglycerol, phosphatidylserine or phosphatidylinositol.
  • negatively charged phospholipids such as phosphatidylglycerol, also serve to prevent spontaneous liposome aggregating, and thus minimize the risk of undersized liposomal aggregate formation.
  • Membrane- rigidifying agents such as sphingomyelin or a saturated neutral phospholipid, at a concentration of at least about 50 mole percent, and 5-15 mole percent of monosialylganglioside, can provide increased circulation of the liposome preparation in the bloodstream, as generally described in U.S. Pat. No. 4,837,028, incorporated herein by reference.
  • the liposome suspension can include lipid-protective agents which protect lipids against free-radical and lipid- peroxidative damages on storage. Lipophilic free-radical quenchers, such as ⁇ tocopherol and water-soluble iron-specific chelators, such as ferrioxianine, are preferred.
  • the liposomes can be sized to achieve a desired size range and relatively narrow distribution of liposome sizes.
  • Several techniques are available for sizing liposome to a desired size. One sizing method is described in U.S. Pat. No. 4,737,323.
  • the liposome suspension is brought to a desired concentration for use in intravenous administration. This can involve re-suspending the liposomes in a suitable volume of injection medium, where the liposomes have been concentrated, for example by centrifugation or ultrafiltration, or concentrating the suspension, where the drug removal step has increased total suspension volume.
  • the suspension is then sterilized by filtration and the liposomes can be administered parenterally or locally in a dose which varies according to, e.g., the manner of administration, the drug being delivered, the particular disease being treated, etc.
  • the dose will vary according to, e.g., the particular agent, the manner of administration, the particular disease being treated and its severity, the overall health and condition of the patient, and the judgment of the prescribing physician.
  • compositions are intended for parenteral, topical, oral or local administration, such as by aerosol or transdermally, for prophylactic and/or therapeutic treatment.
  • the pharmaceutical compositions can be administered in a variety of unit dosage forms depending upon the method of administration.
  • unit dosage forms suitable for oral administration include powder, tablets, pills, and capsules.
  • compositions for intravenous administration which comprise a solution of the agent dissolved or suspended in an acceptable carrier, preferably an aqueous carrier.
  • an acceptable carrier preferably an aqueous carrier.
  • aqueous carriers can be used, e.g., water, buffered water, 0.4% saline, and the like.
  • PBS phosphate buffered saline
  • These compositions can be sterilized by conventional, well-known sterilization techniques, or can be sterile filtered.
  • the resulting aqueous solutions can be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous solution prior to administration.
  • compositions can contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.
  • auxiliary substances such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.
  • nontoxic solid carriers can be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.
  • a pharmaceutically acceptable nontoxic composition is formed by incorporating any of the normally employed excipients, such as those carriers previously listed, and generally 10-95% of active ingredient.
  • the complexes are preferably supplied in finely divided form along with a surfactant and propellant.
  • the surfactant must, of course, be nontoxic, and preferably soluble in the propellant.
  • Representative of such agents are the esters or partial esters of fatty acids containing from 6 to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride such as, for example, ethylene glycol, glycerol, erythritol, arabitol, mannitol, sorbitol, the hexitol anhydrides derived from sorbitol, and the polyoxyethylene and polyoxypropylene derivatives of these esters.
  • the surfactant can constitute 0.1%-20% by weight of the composition, preferably 0.25-5%.
  • the balance of the composition is ordinarily propellant.
  • Liquefied propellants are typically gases at ambient conditions, and are condensed under pressure.
  • suitable liquefied propellants are the lower alkanes containing up to 5 carbons, such as butane and propane; and preferably fluorinated or fluorochlorinated alkanes. Mixtures of the above can also be employed.
  • a container equipped with a suitable valve is filled with the appropriate propellant, containing the finely divided compounds and surfactant. The ingredients are thus maintained at an elevated pressure until released by action of the valve.
  • the agents are formulated into ointments, creams, salves, gels as is generally known in the art. See, -Re-m ⁇ ngt ⁇ n's Pharmaceutical Sciences, supra .
  • compositions are administered to an individual in an amount sufficient inhibit growth of a malignant cell.
  • An amount adequate to accomplish this is defined as "therapeutically effective dose.” Amounts effective for this use will depend on the severity of the disease and the weight and general state of the patient. The following Example is intended to illustrate but not limit the invention.
  • MIF is induced by two growth factors whose effects are signalled through tyrosine kinase receptor pathways (A. Lanahan et al. (1992) Mol . Cell . Biol . 12:3919; A. Ullrich and J. Schlessinger (1990) Cell 61:203) .
  • tyrosine kinase receptor pathways A. Lanahan et al. (1992) Mol . Cell . Biol . 12:3919; A. Ullrich and J. Schlessinger (1990) Cell 61:203.
  • TGF/31 another growth factor which is mitogenic in these cells and whose effect is signalled through a serine/threonine kinase system
  • NIH 3T3 cells were grown serum-free (control) or in the presence of either 10 ng/ml TGF/31 overnight, or 5 ng/ml PDGF for 6 hours.
  • a recombinant MIF gene promoter construct was used to determine whether the induction of MIF mRNA occurs by increased transcription in growth factor stimulated cells.
  • the human MIF gene promoter (V. Paralkar and G. Wistow (1994) Genomics 19:48) was cloned upstream of the CAT (chloramphenicol acetyltransferase) reporter gene and stably integrated into NIH 3T3 cells.
  • the human MIF promoter (1075 to +40) was ligated into the Hindlll site of pSVOATCAT vector (S. Lok et al. (1989) Nucleic Acids Res . 17:3563) and transfected by standard calcium phosphate precipitation (C.
  • CAT activity was measured and expressed as activity/min/ ⁇ g protein. Activity in the absence of serum was used to normalize the activity obtained during various treatments.
  • oligodeoxynucleotides were directly administered to the cell culture medium, an approach which has previously proved successful for suppression of interleukin-lo (J.A. Maier et al. (1990) Science 249:1570).
  • oligos were used: aMIFl: GATGAACATA GGCATGGTGG CGGAGAGACT (SEQ ID NO:3) sMIFl: AGTCTCTCCG CCACCATGCC TATGTTCATC (SEQ ID NO:4) lFIMa: TCAGAGAGGC GGTGGTACGG ATACAAGTAG (SEQ ID NO:5) aMIF2: CTCTTATAAA CCATTTATTT CTCCCGGCTG (SEQ ID NO:6).
  • Oligonucleotide aMIFl is an antisense to mouse MIF mRNA (A.
  • Oligonucleotide sMIFl is the complement of aMIFl as a sense control.
  • Oligonucleotide lFIMa is the reverse of aMIFl as a control for composition.
  • Oligonucleotide aMIF2 is a second antisense oligo with a similar G+C/A+T ratio to aMIFl, targeted to the polyadenylation signal of mouse MIF.
  • NIH 3T3 cells were grown in the presence of these oligos and treated with growth factors as before (Fig. 3A) .
  • NIH 3T3 cells in 0.2% serum at a density of lxlO 5 per ml were seeded in 24 well tissue culture plates at 0.5 ml per well and treated with growth factors and oligos as indicated. After 22 hours, 3 H-thymidine was added to the cells and incorporation into DNA was measured for two hours (D. Danielpour et al. (1989) J. Cell . Physiol . 138:79). For recovery of cells from aMIFl treatment, aMIFl-containing medium was replaced by medium containing 10% serum after 22 hours. After a further 22 hours, cells were labelled with 3 H-thymidine as before.
  • cyclin-E is a marker for late GI and is a rate-limiting factor for the Gl/S transition
  • cyclin-A is a marker for S phase
  • G2 is a marker for cells in G2 (M. Ohtsubo and J.M. Roberts (1993) Science 259:1908; C.J. Sherr (1993) Cell 73:1059).
  • Cells were grown with or without aMIFl as in Fig. 3.
  • Cell extracts were examined by western blotting with rabbit anti-human cyclin A, B and E antibodies (UBI, Lake Placid, NY) as described above.
  • UBI rabbit anti-human cyclin A, B and E antibodies
  • PDGF-treated NIH 3T3 cells in the presence of aMIFl cyclin-B and cyclin-A were greatly reduced while cyclin-E was still detectable (Fig. 4A) . This is consistent with cell cycle arrest in GI and suggests that MIF is required for entry into S phase.
  • MIF-constitutive NIH 3T3 cells had significantly higher rates of proliferation (Fig. 5) than controls and contained diminished populations in GI relative to S and G2/M (Fig. 5) .
  • MIF act as positive regulatory subunits of important cell cycle control systems. Since MIF acts late in Gl, candidate targets for such regulatory activity are cyclin-E and its partner CDK2 which are essential for Gl/S transition and Rb, the product of the retinoblastoma gene, which is an important inhibitor of Gl/S transition (R.E. Hollingsworth, Jr., et al (1993) Curr . Opin . Genet. £»ev. 3:55).
  • NIH 3T3 cells grown in the presence of 10% serum were lysed as an incubated in binding buffer (25 mM Tris pH 7.4, 50 mM NaCl, 0.5% Sodium deoxycholate, 0.2% NP-40, 0.2% SDS, 1 mM PMSF, 50 ⁇ g/ml aproteinin, 50 ⁇ M leupeptin) with either agarose conjugated anti-Rb (Santa Cruz Biotech) , or CDK2 (UBI) overnight, or with anti-cyclin E (UBI) for 1 hour followed by addition of protein A agarose and overnight incubation.
  • binding buffer 25 mM Tris pH 7.4, 50 mM NaCl, 0.5% Sodium deoxycholate, 0.2% NP-40, 0.2% SDS, 1 mM PMSF, 50 ⁇ g/ml aproteinin, 50 ⁇ M leupeptin
  • Agarose beads were then washed three times in binding buffer and boiled in the presence of SDS PAGE sample buffer to release bound proteins, followed by SDS PAGE and Western blot analysis using ECL detection as above.
  • Some rabbit immunoglobulin-derived material was released from the beads and subsequently detected in all lanes by horse-radish peroxidase linked to goat anti-rabbit immunoglobulin (Vector Labs) .
  • the effect of antisense oligonucleotides on the growth of cancer cells also was tested.
  • the proliferation of cultured cells transformed with ras, raf , or sis oncogenes was essentially halted when the cells were exposed to aMIFl.

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Abstract

Cette invention se rapporte à des procédés d'inhibition de la prolifération cellulaire par inhibition de l'activité mitogène du facteur inhibiteur de la migration des macrophages ('MIF'). Des acides nucléiques inhibiteurs dirigés contre le MIF inhibent la prolifération cellulaire à la fois dans des cellules induites par des facteurs de croissance et dans des cellules transformées (malignes) exprimant des oncogènes.
PCT/US1995/015000 1994-11-16 1995-11-16 Procedes d'inhibition de la proliferation cellulaire par inhibition de l'activite mitogene du facteur inhibiteur de la migration des macrophages WO1996015242A2 (fr)

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999042578A2 (fr) * 1998-01-30 1999-08-26 Cold Spring Harbor Laboratory Modulation de la proliferation cellulaire, procedes et reactifs
WO2000006189A2 (fr) * 1998-07-27 2000-02-10 The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Conversion de quinones naturelles en derives de quinones au moyen du facteur d'inhibition de migration de macrophages et procedes d'utilisation correspondants
WO2001053317A1 (fr) * 2000-01-20 2001-07-26 Isis Pharmaceuticals, Inc. Modulation antisens d'expression du facteur inhibiteur de migration du macrophage
US7084141B2 (en) 2001-05-24 2006-08-01 Avanir Pharmaceuticals Inhibitors of macrophase migration inhibitory factor and methods for identifying the same
US7235546B2 (en) 2003-02-14 2007-06-26 Avanir Pharmaceuticals Inhibitors of macrophage migration inhibitory factor and methods for identifying the same
US7365200B2 (en) 2005-03-24 2008-04-29 Avanir Pharmaceuticals Thienopyridinone derivatives as macrophage migration inhibitory factor inhibitors
JP2009155335A (ja) * 1996-10-24 2009-07-16 Cytokine Pharmasciences Inc 抗癌療法のためのマクロファージマイグレーション阻害因子アンタゴニストの使用
US9958456B2 (en) 2011-10-07 2018-05-01 Baxalta Incorporated OxMIF as a diagnostic marker

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994026307A1 (fr) * 1993-05-17 1994-11-24 The Picower Institute For Medical Research Inhibition de la migration du facteur inhibiteur dans le traitement de maladies impliquant une toxicite transmise par la cytokine

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994026307A1 (fr) * 1993-05-17 1994-11-24 The Picower Institute For Medical Research Inhibition de la migration du facteur inhibiteur dans le traitement de maladies impliquant une toxicite transmise par la cytokine

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ANNUAL MEETING OF THE AMERICAN SOCIETY FOR BIOCHEMISTRY AND MOLECULAR BIOLOGY, SAN FRANCISCO, CALIFORNIA, USA, MAY 21-25, 1995. & FASEB JOURNAL 9 (6). 1995. A1332, XP002002563 PARALKAR, V. ET AL.: "MIF: An essential intermediate for growth factor induced mitogenesis." *
GENOMICS, (1994 JAN 1) 19 (1) 48-51, XP000569580 PARALKAR, V. ET AL.: "Cloning the human gene for macrophage migration inhibitory factor ( MIF )." cited in the application *

Cited By (27)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2009155335A (ja) * 1996-10-24 2009-07-16 Cytokine Pharmasciences Inc 抗癌療法のためのマクロファージマイグレーション阻害因子アンタゴニストの使用
WO1999042578A2 (fr) * 1998-01-30 1999-08-26 Cold Spring Harbor Laboratory Modulation de la proliferation cellulaire, procedes et reactifs
WO1999042578A3 (fr) * 1998-01-30 1999-12-23 Cold Spring Harbor Lab Modulation de la proliferation cellulaire, procedes et reactifs
WO2000006189A2 (fr) * 1998-07-27 2000-02-10 The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Conversion de quinones naturelles en derives de quinones au moyen du facteur d'inhibition de migration de macrophages et procedes d'utilisation correspondants
WO2000006189A3 (fr) * 1998-07-27 2000-05-11 Us Health Conversion de quinones naturelles en derives de quinones au moyen du facteur d'inhibition de migration de macrophages et procedes d'utilisation correspondants
WO2001053317A1 (fr) * 2000-01-20 2001-07-26 Isis Pharmaceuticals, Inc. Modulation antisens d'expression du facteur inhibiteur de migration du macrophage
US6268151B1 (en) * 2000-01-20 2001-07-31 Isis Pharmaceuticals, Inc. Antisense modulation of macrophage migration inhibitory factor expression
AU2001229537B2 (en) * 2000-01-20 2004-06-03 Isis Pharmaceuticals, Inc. Antisense modulation of macrophage migration inhibitory factor expression
US7192961B2 (en) 2001-05-24 2007-03-20 Avanir Pharmaceuticals Inhibitors of macrophage migration inhibitory factor and methods for identifying the same
US7238809B2 (en) 2001-05-24 2007-07-03 Avanir Pharmaceuticals Process for the preparation of inhibitors of macrophage migration inhibitory factor
US7157469B2 (en) 2001-05-24 2007-01-02 Avanir Pharmaceuticals Inhibitors of macrophage migration inhibitory factor and methods for identifying the same
US7192955B2 (en) 2001-05-24 2007-03-20 Avanir Pharmaceuticals Inhibitors of macrophage migration inhibitory factor and methods for identifying the same
US7105519B2 (en) 2001-05-24 2006-09-12 Avanir Pharmaceuticals Inhibitors of macrophage migration inhibitory factor and methods for identifying the same
US7202248B2 (en) 2001-05-24 2007-04-10 Avanir Pharmaceuticals Inhibitors of macrophage migration inhibitory factor and methods for identifying the same
US7230106B2 (en) 2001-05-24 2007-06-12 Avanir Pharmaceuticals Process for the preparation of inhibitors of macrophage migration inhibitory factor
US7732146B2 (en) 2001-05-24 2010-06-08 Avanir Pharmaceuticals Method for screening an agent that modulates activity of macrophage migration inhibitory factor
US7235565B2 (en) 2001-05-24 2007-06-26 Avanir Pharmaceuticals Inhibitors of macrophage migration inhibitory factor and methods for identifying the same
US7129236B2 (en) 2001-05-24 2006-10-31 Avanir Pharmaceuticals Inhibitors of macrophage migration inhibitory factor and methods for identifying the same
US7084141B2 (en) 2001-05-24 2006-08-01 Avanir Pharmaceuticals Inhibitors of macrophase migration inhibitory factor and methods for identifying the same
US7514225B2 (en) 2001-05-24 2009-04-07 Avanir Pharmaceuticals Method for screening an agent that modulates activity of macrophage migration inhibitory factor
US7435737B2 (en) 2001-05-24 2008-10-14 Avanir Pharmaceutials Inhibitors of macrophage migration inhibitory factor and methods for identifying the same
US7432374B2 (en) 2001-05-24 2008-10-07 Avanir Pharmaceuticals Inhibitors of macrophage migration inhibitory factor and methods for identifying the same
US7312221B2 (en) 2003-02-14 2007-12-25 Avanir Pharmaceuticals Inhibitors of macrophage migration inhibitory factor and methods for identifying the same
US7312220B2 (en) 2003-02-14 2007-12-25 Avanir Pharmaceuticals Inhibitors of macrophage migration inhibitory factor and methods for identifying the same
US7235546B2 (en) 2003-02-14 2007-06-26 Avanir Pharmaceuticals Inhibitors of macrophage migration inhibitory factor and methods for identifying the same
US7365200B2 (en) 2005-03-24 2008-04-29 Avanir Pharmaceuticals Thienopyridinone derivatives as macrophage migration inhibitory factor inhibitors
US9958456B2 (en) 2011-10-07 2018-05-01 Baxalta Incorporated OxMIF as a diagnostic marker

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