EP1210432A2 - In vivo behandlung von gelenkskrankheiten durch verwendung von interleukin-1 rezeptor-antagonisten - Google Patents

In vivo behandlung von gelenkskrankheiten durch verwendung von interleukin-1 rezeptor-antagonisten

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Publication number
EP1210432A2
EP1210432A2 EP00992198A EP00992198A EP1210432A2 EP 1210432 A2 EP1210432 A2 EP 1210432A2 EP 00992198 A EP00992198 A EP 00992198A EP 00992198 A EP00992198 A EP 00992198A EP 1210432 A2 EP1210432 A2 EP 1210432A2
Authority
EP
European Patent Office
Prior art keywords
joint
mammal
viral particles
interleukin
receptor antagonist
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00992198A
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English (en)
French (fr)
Inventor
David D. Frisbie
Wayne C. Mcillwraith
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Colorado State University Research Foundation
Original Assignee
Colorado State University Research Foundation
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Filing date
Publication date
Application filed by Colorado State University Research Foundation filed Critical Colorado State University Research Foundation
Publication of EP1210432A2 publication Critical patent/EP1210432A2/de
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner

Definitions

  • the invention relates to the in vivo treatment of joint disease using gene therapy.
  • in vivo administration of vectors encoding interleukin-1 receptor antagonists is disclosed.
  • Joint disease is a severe mobility and career modifying disease in man and horses (Robert Davis, Horses give a leg up to human knee research, U.S.A. Today, April 6, 1999, at 10D).
  • the etiopathogenesis of joint disease is not clearly understood, however, may mediators that lead to degeneration within the joint environment have been identified.
  • Cytokines especially interleukin-1 and tumor necrosis factor, have been defined as initiating mediators of a degenerative cascade that propagates joint disease.
  • anti-cytokine proteins it has been demonstrated that blocking these molecules can significantly alter the course of joint disease.
  • Non- viral vectors typically refer to synthetic molecules that facilitate the uptake of DNA into cells by condensing the DNA with lipids, peptides, proteins, inactivated virus particles, crystals of calcium phosphate, or coated microprojectiles.
  • Viral vectors are viruses from which the viral genes have been removed to allow insertion of the therapeutic gene(s), and the viral/vector has usually been rendered incapable of replicative spread. As a general rule viral vectors give much higher efficiencies of gene delivery than non-viral vectors. Many well characterized viruses have been explored for use as vectors, but to date retroviral and adenoviral vectors have proven the most useful. (Verma, J.M. and Somia, N., Nature 389: 239-242, 1997; Martin, P.A. and Thomas, S.M., Hum. Gene Ther., 9: 87-114, 1998; Robbins, P.D., et al., Trends Biotech., 16: 35-40, 1998).
  • Retroviral vectors integrate their DNA (including a gene sequence of interest) into the chromosomal DNA of the target cells, ensuring gene transfer to the target cell progeny.
  • retroviral vectors only transduce (infect) dividing cells, making them useful in certain situations but less useful in others. If for example the target population of cells were gastrointestinal epithelium which divide very rapidly, a retroviral vector could be quite useful in a gene transfer protocol.
  • Adenoviral vectors can transduce non-dividing cells allowing them to be used on a larger range of cell types. If the goal of a protocol was to transduce slow or non-dividing cells, such as neurons, an adenoviral vector would be a superior choice. (Robbins, P.D., et al., Trends Biotech., 16: 35-40, 1998) In addition to work based on the engineering of equine gene vaccines, (Guo,
  • cytokine modulating agents receptor antagonists, soluble forms of receptors, and monoclonal antibodies against cytokines
  • cytokine modulating agents have shown significant therapeutic benefits (Wood, D.D., et al., Arthritis and Rheumatism, 28: 853-862, 1996; Goodwin, R.G., et al., Mol. Cell Bio , 11 : 3020-3026, 1991; Loetscher, H., et al., Cell, 61: 351-359, 1990; Schall, TJ., et al., Cell, 61: 361-370, 1990; Lewthwaite, J., et al., J.
  • Direct administration of vectors encoding the interleukin-1 receptor antagonist protein (IRAP) to joint tissue is disclosed as a favorable method for treating joint disease.
  • a preferred embodiment involves the intraarticular injection of adenovirus vectors into the joint space of an arthritic horse leading to long term production of the interleukin-1 receptor antagonist protein, and an accompanying improvement in the condition of the joint.
  • Direct administration of vectors encoding the interleukin-1 receptor antagonist protein (IRAP) to joint tissue may also be performed as a preventitive or prophylactic measure.
  • IIRAP interleukin-1 receptor antagonist protein
  • Asterisks (*) denotes a statistical difference (P - value ⁇ 0.05) in the data point compared to placebo treatment at that time period.
  • Asterisks (*) denotes a statistical difference (P - value ⁇ 0.05) in the data point compared to placebo treatment at that time period.
  • Amplification refers to increasing the number of copies of a desired nucleic acid molecule. Amplification routinely refers to the polymerase chain reaction (PCR).
  • Arthrocentesis refers to the addition to or replacement of fluid in a mammalian joint through a needle inserted into the synovial capsule. Arthrocentesis may be performed to remove fluid for therapeutic or analytical purposes, or to add a fluid to the joint.
  • coding sequence refers to the region of continuous sequential nucleic acid triplets encoding a protein, polypeptide, or peptide sequence.
  • Codon refers to a sequence of three nucleotides that specify a particular amino acid.
  • expression refers to the transcription of a gene to produce the corresponding mRNA and translation of this mRNA to produce the corresponding gene product, i.e., a peptide, polypeptide, or protein.
  • Ex vivo refers to preparation of one or more transgenic cells in vitro, followed by administration of the cells in vivo to an animal.
  • gene refers to chromosomal DNA, plasmid DNA, cDNA, synthetic DNA, or other DNA that encodes a peptide, polypeptide, protein, or RNA molecule, and regions flanking the coding sequence involved in the regulation of expression.
  • Identity refers to the degree of similarity between two nucleic acid or protein sequences. An alignment of the two sequences is performed by a suitable computer program. A widely used and accepted computer program for performing sequence alignments is CLUSTALW vl.6 (Thompson, et al. Nucl. Acids Res., 22: 4673-4680, 1994).
  • In vitro refers to in the laboratory.
  • In vivo refers to in a living organism.
  • 'TRAP refers to interleukin-1 receptor antagonist protein.
  • h-IRAP refers to human ⁇ RAP
  • eq- ⁇ RAP refers to equine IRAP
  • adlRAP refers to an adenovirus vector encoding IRAP.
  • Joint disease refers to diseases which restrict the mobility of joints in mammals. Joint disease includes osteoarthritis and rheumatoid arthritis.
  • “Mutation” refers to any change or alteration in the sequence of a gene. Several types exist, including point, frame shift, and splicing. “Nucleic acid” refers to deoxyribonucleic acid (DNA) and ribonucleic acid
  • Open reading frame refers to a region of DNA or RNA encoding a peptide, polypeptide, or protein.
  • “Plasmid” refers to a circular, extrachromosomal. self-replicating piece of DNA.
  • PCR Polymerase chain reaction
  • Restriction enzyme refers to an enzyme that recognizes a specific palindromic sequence of nucleotides in double stranded DNA and cleaves both strands; also called a restriction endonuclease. Cleavage typically occurs within the restriction site, "Vector” refers to a plasmid, cosmid, bacteriophage, or virus that carries foreign DNA into a host organism.
  • the invention is directed generally towards methods to reduce the effects of joint disease in mammals.
  • the method comprises selecting a mammal suspected of having a joint afflicted with a joint disease; administering one or more viral particles to the joint by arthrocentesis; and detecting a reduction in the effects of the joint disease on the treated joint; wherein the viral particles comprise a nucleic acid sequence encoding an equine interleukin-1 receptor antagonist protein.
  • a reduction in the effects of the joint disease on the treated joint may be readily assessed by determining a lameness value between 0 and 5, according to the guidelines established by the American Association of Equine Practitioners (Anonymous Guide for veterinary service and judging of equestrian events.
  • the mammal may generally be any mammal having joints susceptible to joint disease. More preferably, the mammal is a horse, rabbit, mouse, dog, cow, donkey, mule, or human. Most preferably, the mammal is a horse or human.
  • a sufficient quantity of viral particles are administered to achieve a reduction in the effects of joint disease, and preferably at least about 10 10 viral particles are administered to the joint.
  • the joint disease is preferably rheumatoid or osteoarthritis.
  • the viral particles are preferably adenoviral particles or retroviral particles, and more preferably are adenoviral particles.
  • the nucleic acid sequence encoding an equine interleukin-1 receptor antagonist protein preferably encodes SEQ ID NO:5, hybridizes to the reverse complement of SEQ ID NO:4 under stringent hybridization conditions, or is at least 95% identical to SEQ ID NO:4, and more preferably is SEQ ID NO:4.
  • the invention is directed towards a method to produce interleukin-1 receptor antagonist protein, the method comprising: selecting a mammal comprising a joint for treatment; and administering one or more viral particles to the joint by arthrocentesis; wherein: the viral particles comprise a nucleic acid sequence encoding an equine interleukin-1 receptor antagonist protein; and the concentration of interleukin-1 receptor antagonist protein in the administered joint is higher than the concentration of interleukin-1 receptor antagonist protein an unadministered joint.
  • the concentration of interleukin-1 receptor antagonist protein is preferably measured using a commercially available kit and manufacture's recommendation (QuantikineTM Human IL-lra immunoassay, R & D Systems, Minneapolis, MN).
  • the concentration of equine interleukin-1 receptor antagonist protein is directly measured; in a horse, the production of interleukin-1 receptor antagonist protein is demonstrated by an increase in concentration over the concentration in a non- treated joint.
  • the mammal may generally be any mammal having joints susceptible to joint disease. More preferably, the mammal is a horse, rabbit, mouse, dog, cow, donkey, mule, or human. Most preferably, the mammal is a horse or human.
  • a sufficient quantity of viral particles are administered to produce interleukin-1 receptor antagonist protein, and preferably at least about 10 10 viral particles are administered to the joint.
  • the viral particles are preferably adenoviral particles or retroviral particles, and more preferably are adenoviral particles.
  • the nucleic acid sequence encoding an equine interleukin-1 receptor antagonist protein preferably encodes SEQ ID NO:5, hybridizes to the reverse complement of SEQ ID NO:4 under stringent hybridization conditions, or is at least 95% identical to SEQ ID NO:4, and more preferably is SEQ ID NO:4.
  • the invention is further useful in a preventitive or prophylactic role in delaying or preventing the onset of joint disease in a mammal.
  • This embodiment is directed towards a method to delay or prevent the onset of joint disease in a mammal, the method comprising: selecting a mammal comprising a joint for treatment; and administering one or more viral particles to the joint by arthrocentesis; wherein the viral particles comprise a nucleic acid sequence encoding an equine interleukin-1 receptor antagonist protein.
  • the mammal may generally be any mammal having joints susceptible to joint disease. More preferably, the mammal is a horse, rabbit, mouse, dog, cow, donkey, mule, or human. Most preferably, the mammal is a horse or human.
  • a sufficient quantity of viral particles are administered to delay or prevent the onset of joint disease, and preferably at least about 10 10 viral particles are administered to the joint.
  • the viral particles are preferably adenoviral particles or retroviral particles, and more preferably are adenoviral particles.
  • the nucleic acid sequence encoding an equine interleukin-1 receptor antagonist protein preferably encodes SEQ ID NO:5, hybridizes to the reverse complement of SEQ ID NO:4 under stringent hybridization conditions, or is at least 95% identical to SEQ ID NO:4, and more preferably is SEQ ID NO:4.
  • An additional alternative embodiment is directed towards a recombinant equine synoviocyte cell comprising a structural nucleic acid sequence encoding an equine interleukin-1 receptor antagonist protein, wherein the copy number of the structural nucleic acid sequence in the recombinant equine synoviocyte cell is higher than the copy number of the structural nucleic acid sequence in a wild type equine synoviocyte cell. Copy number of a nucleic acid sequence may be determined by Southern blotting, or by PCR.
  • a recombinant equine synoviocyte cell comprising a structural nucleic acid sequence encoding an equine interleukin-1 receptor antagonist protein may be administered to a mammalian joint in an ex vivo treatment method.
  • the ex vivo treatment method may be used to reduce the effects of joint disease, or may be used in a preventitive or prophylactic role.
  • the mammal may generally be any mammal having joints susceptible to joint disease. More preferably, the mammal is a horse, rabbit, mouse, dog, cow, donkey, mule, or human. Most preferably, the mammal is a horse or human.
  • the Examples below use the horse and horse cells as a model system for the development and validation of this invention.
  • the horse is an athletic species with naturally occurring joint disease, and the equine nucleic acid sequence for IRAP is known.
  • Other mammals including rabbit, mouse, dog, cow, donkey, mule and humans, are predicted to benefit from the ERAP gene therapy strategy due to their similarities in joint structure to the horse.
  • Synovium was aseptically harvested from grossly normal metacarpophalangeal joints of 5 horses ranging in age from 10 - 15 years. During processing synovium was stored in phosphate buffered saline (PBS) at 4°C unless otherwise noted. Synovium was further divided into 3 mm 2 pieces and placed in supplemented Ham's F12 media with 0.2% collagenase and cultured for 4 hours in a 37°C, 5% CO 2 environment with gentle agitation.
  • PBS phosphate buffered saline
  • the appearance of the synovial fluid is noted by looking at the color and turbidity in a glass test tube. Color is rated as straw, yellow, orange, or red. Turbidity is qualitatively rated as clear, cloudy, or opaque. If iatrogenic blood contamination is observed, the sample is centrifuged for 10 minutes at 3000 rpm. This will separate the blood from the synovial fluid.
  • the slides are labeled with a permanent marker: horse nu her, joint location, cytospin, and the date.
  • the slide, cytospin funnel and the funnel holder are put together and 2-5 drops of hyaluronidase treated fluid is added to the funnel.
  • the funnels are placed in the cytospin rotor and then the samples are spun for 4 minutes in the cytocentrifuge. Then slides are removed from the holders and are allowed to dry. A small circle of cells will be in the middle of the slide. When dry, the slides are stained with the dif-quick automatic stainer. Microscopic Cvtospin Slide Interpretation
  • a 100 cell differential of neutrophils, large mononuclear cells, lymphocytes and eosinophils is performed determining a breakdown of these four types of cells. If there are not 100 cells to count, a percentage for each type ofcell counted is calculated based on the total number of cells counted.
  • a hematocrit tube is filled with hyaluronidase treated or non-treated synovial fluid by capillary action.
  • the fluid is then placed on the refractometer to read the specific gravity.
  • the total protein is calculated from the specific gravity according to the following table.
  • synovial fluid Several drops of synovial fluid are added to 20 ml of 5% acetic acid in a glass beaker. After 1 minute, the beaker is gently swished in a circular motion. The clump of synovial fluid can then he evaluated as good, fair or poor by dipping a wooden stir stick into the mixture and evaluating the clump by how well it holds together: Good - tight, ropy clump - holds together completely; Fair - partially breaks down at the edges; Poor - flakes and shreds - does not stay together.
  • the adenoviral vector backbone used in this study were replication-deficient type 5 adenovirus lacking El and E3 loci. (Yeh, P. and Perricaudet, M., FASEB J, 11: 615-623, 1997). Construction of adenovirus vectors is well known in the art (see Hardy, S. et al., J. Virol. 71: 1842-1849, 1997). The coding region for equine IRAP (Howard, R.D., et al., Am. J. Vet.
  • Rest ⁇ ction endonuclease BamHI sites were added to the IRAP coding sequence by polymerase chain reaction (PCR) using oligonucleotide p ⁇ mers Horse3 (SEQ ID NO:l) and Horse5 (SEQ ID NO:2).
  • the coding region of the equine ERAP sequence is from nucleotides 14-548 of SEQ ED NO:3.
  • PCR amplification produced SEQ ID NO:4.
  • the amplified product was purified from a 1.5% agarose gel and hgated into the pAdlox vector (Somatix, Alameda, CA, GenBank Accession No. U62024).
  • the resulting plasmid was digested with rest ⁇ ction enzyme Sfil, and used in a co-transfection with the psi5 adenoviral backbone (Somatix Therapy Corp., Alameda, CA) into CRE8 cells. Plaques were isolated, expanded, and characterized for insertion of the equine IRAP cDNA and its expression. To generate stocks of virus, confluent flasks of CRE8 cells were infe ' cted with the ad.eq-ERAP virus. After detection of significant cytopathic effects, the cells were harvested, pelleted, resuspended in 5 mL of saline and stored at -80°C.
  • the cell pellet was first lysed by three rounds of freeze-thaw. The cell debris was pelleted by centnfugation, and the cleared lysate collected. Virus was banded three times over successive cesium chlo ⁇ de step gradients. The virus was collected after dialysis, aliquotted, and stored at -80°C.
  • EXAMPLE 6 Lac-Z. IRAP or PGE2 detection
  • X-gal (5- bromo-4-chloromdolyl- ⁇ -D-galactose).
  • X-gal turns a blue color when in the presence of ⁇ -galactosidase and therefore can be used as a marker of transgene production.
  • Neat media aliquots were used for determination of IRAP utilizing a commercially available kit and manufacture's recommendation (QuantikineTM Human IL-lra immunoassay, R & D Systems, Minneapolis, MN). Neat media aliquots were used for determination of PGE 2 utilizing a commercially available kit and manufacture's recommendation (TiterZyme® PGE 2 enzyme immunoassay kit, PerSeptive Biosystems, Inc., Framingham, MA).
  • Two flasks of synoviocytes were transduced with the adLacZ vector at a multiplicity of infection (MOI) of 0, 1, 10 and 100 to determine transduction frequencies.
  • MOI multiplicity of infection
  • Two flasks of synoviocytes were infected with eq-adlRAP at 0, 1, 10, and 100 MOI to determine the concentration of ERAP produced. After transductions the cells were cultured for an additional 48 hours after which time the media was removed and stored (-80°C) and the cell numbers in each flask were estimated using a hemocytometer.
  • the concentrations of ERAP in the media samples were normalized to 1 x 10 6 cells and a 48-hour culture period.
  • Synoviocytes were either non-transduced or transduced (eq-adlRAP) at 10 MOI, cultured for 2 days, and then lOng of human recombinant II- l ⁇ (Life Technologies, Gaithersburg, MD, USA) was added to the media of some cells followed by an additional 2 days of culture. The media was collected and stored (- 80°C) for IRAP and PGE 2 determination as well as estimations of cell numbers in each flask.
  • the diluent was chosen as the placebo to test the antigenicity of both the vector and proteins produced in association with its presence.
  • Each viral dose (1 x 10 9 , 1 x 10 10 , 1 x 10", 2 x 10", and 5 x 10" particles/joint) was tested in two different horses.
  • the transduced and placebo joints were aseptically prepared and arthrocentesis performed using a 12 mL syringe and a 20 ga x 1.5" (5.08 cm) needle.
  • Approximately 4 mL of synovial fluid was collected for routine synovial fluid analysis (total protein, WBC counts, differential cell count, color estimation, and quality of mucin clot) and IRAP quantification.
  • synovial fluid samples were split into tubes containing EDTA (routine synovial fluid analysis) or sodium citrate (IRAP determination). During the same procedure either the vector or placebo treatment was administered directly into the joint cavity. Arthrocenteses were repeated on days 3, 7 and at 7 day intervals thereafter until day 35 post transduction or until measured ERAP protein was similar to control levels.
  • Modification and changes may be made in the sequence of the IRAP protein used in the present invention and the encoding nucleic acid sequences and still obtain a functional molecule that encodes a protein with desirable properties.
  • the following is a discussion based upon changing the ammo acid sequence of a protein to create an equivalent, or possibly an improved, second-generation molecule.
  • the amino acid changes may be achieved by changing the codons of the nucleic acid sequence, according to the codons given in Table 3.
  • Certain amino acids may be substituted for other amino acids in a protein sequence without appreciable loss of enzymatic activity. It is thus contemplated that various changes may be made in the peptide sequences of the disclosed protein sequences, or their corresponding nucleic acid sequences without appreciable loss of the biological activity. In making such changes, the hydropathic index of amino acids may be considered.
  • the importance of the hydropathic amino acid index in conferring interactive biological function on a protein is generally understood in the art (Kyte and Doolittle, J. Mol Biol., 157: 105-132, 1982). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.
  • Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics. These are: isoleucine (+4.5); valine (+4.2) leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9) alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9) tyrosine (-1-3); proline (-1-6); histidine (-3.2) glutamate/glutamine/aspartate/asparagine (-3.5); lysine (-3.9); and arginine (-4.5).
  • amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e., still obtain a biologically functional protein.
  • substitution of amino acids whose hydropathic indices are within ⁇ 2 is preferred, those within ⁇ 1 are more preferred, and those within ⁇ 0.5 are most preferred.
  • hydrophilicity values have been assigned to amino acids: arginine/lysine (+3.0); aspartate/glutamate (+3.0 ⁇ 1); serine (+0.3); asparagine/glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ⁇ 1); alanine/histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine/isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); and tryptophan (-3.4).
  • amino acid may be substituted by another amino acid having a similar hydrophilicity score and still result in a protein with similar biological activity, i.e., still obtain a biologically functional protein.
  • substitution of amino acids whose hydropathic indices are within ⁇ 2 is preferred, those within ⁇ 1 are more preferred, and those within ⁇ 0.5 are most preferred.
  • amino acid substitutions are therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
  • substitutions which take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine, and isoleucine. Changes which are not expected to be advantageous may also be used if these resulted in functional proteins.
  • compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention.

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  • Health & Medical Sciences (AREA)
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EP00992198A 1999-09-07 2000-09-06 In vivo behandlung von gelenkskrankheiten durch verwendung von interleukin-1 rezeptor-antagonisten Withdrawn EP1210432A2 (de)

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US15270799P 1999-09-07 1999-09-07
US152707P 1999-09-07
PCT/US2000/040823 WO2001025435A2 (en) 1999-09-07 2000-09-06 In vivo treatment of joint disease using interleukin-1 receptor antagonists

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RS57852B1 (sr) 2004-04-02 2018-12-31 Swedish Orphan Biovitrum Ab Publ Postupci za smanjenje agregacije il-1ra
WO2020112853A1 (en) 2018-11-28 2020-06-04 Genascence Corporation Methods and compositions for treating osteoarthritis

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US5747072A (en) * 1993-07-30 1998-05-05 University Of Michigan Adenoviral-mediated gene transfer to synovial cells in vivo
JPH1077298A (ja) * 1996-09-05 1998-03-24 Nippon Seibutsu Kagaku Kenkyusho 馬インターロイキン−１レセプターアンタゴニスト

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