WO2001007085A1 - Nouveaux procedes et reactifs convenant au traitement de l'arthrose - Google Patents

Nouveaux procedes et reactifs convenant au traitement de l'arthrose Download PDF

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Publication number
WO2001007085A1
WO2001007085A1 PCT/US2000/018938 US0018938W WO0107085A1 WO 2001007085 A1 WO2001007085 A1 WO 2001007085A1 US 0018938 W US0018938 W US 0018938W WO 0107085 A1 WO0107085 A1 WO 0107085A1
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Prior art keywords
wisp3
protein
joint
family
growth factor
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PCT/US2000/018938
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English (en)
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Matthew L. Warman
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Case Western Reserve University
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Priority to AU60889/00A priority Critical patent/AU6088900A/en
Publication of WO2001007085A1 publication Critical patent/WO2001007085A1/fr

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    • 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
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals

Definitions

  • This invention generally relates to novel methods for screening suspected therapeutic compounds for the treatment of progressive pseudorheumatoid dysplasia
  • PPD PFD
  • OA Osteoarthritis
  • Osteoarthritis is a degenerative disorder of joints and cartilage.
  • the articular surfaces are disrupted involving a loss of normal collagen architecture and a chondrocyte response that replaces the abnormal structure.
  • the replacement cartilage is less resistant to wear than the original and the progression of OA eventually results in a complete loss of any articular joint protection by the extracellular matrix.
  • OA hereditary nature of OA was first reported in the 1940's.
  • the most genetically susceptible constituents of cartilage function in OA include; 1) the functional organization of macromolecular elements of the cartilage determined by specific associations between proteins, proteoglycans, and cells, 2) alterations of collagen and proteoglycan side-chains which are responsible for the structural integrity of the joint, and 3) proteins involved in intracellular signalling processes which affect chondrocyte synthesis and catabolism of matrix components.
  • Genetic models linked to OA have focused on the fibrillar collagens of types II, V, and XI. Of these reports, mutations in type II collagen are most common. These single point mutations usually involve a C to T substitution and effect an obligatory glycine.
  • OA joint pain related to physical activity.
  • NSAIDs non-steroidal anti- inflammatory drugs
  • the available palliative effects from NSAIDs do not provide adequate pain relief or amelioration of other symptoms thus stimulating the development of alternative treatments.
  • HA hyaluronic acid
  • HA is able to modulate a variety of cellular functions, suppress the activities of pro-inflammatory mediators, or attenuate nociceptive responses.
  • recent studies with animal models of noninflammatory OA have questioned the ability of HA to protect articular cartilage degeneration directly.
  • HA is implicated in the efficacy of glucosamine administration to OA patients.
  • the traditional explanation of glucosamine therapy is that it promotes the synthesis of cartilage proteoglycans.
  • glucosamine stimulates synovial production of HA.
  • This invention generally relates to novel compounds that may be used as lubricants of tissue and joints. Additionally, the present invention provides reagents for the screening of compounds that may be used as therapeutic agents in the treatment of Osteoarthritis.
  • the present invention contemplates the Wisp3 protein, or portions thereof, in a preparation suitable for use as a lubricant.
  • the present invention contemplates that such a preparation can be used in a method of treatment.
  • the method comprises a) providing: i) a subject (e.g. a human or animal), and ii) a preparation comprising the Wisp3 protein, or portion thereof; and b) administering said preparation to said subject to lubricate the subjects tissue or joints.
  • the method comprises a) providing: i) a subject (e.g. a human or animal) diagnosed with arthritis, and ii) a preparation comprising the Wisp3 protein, or portion thereof; and b) administering said preparation to said subject.
  • the method comprises a) providing: i) a subject (e.g. a human or animal) with symptoms of osteoarthritis, and ii) a preparation comprising the Wisp3 protein, or portion thereof; and b) administering said preparation to said subject under conditions such that said symptoms (e.g. joint pain, loss of range of movement, joint damage, etc.) are reduced.
  • the preparation can have other ingredients.
  • said preparation further comprises a local anesthetic.
  • the present invention contemplates a composition, comprising Wisp3 protein, or portion thereof, in combination with an anesthetic.
  • said administering comprises intra-articular injection.
  • said administering comprises intravenous injection.
  • said preparation is administered topically.
  • Such topically administered preparations may have ingredients that permit penetration of the skin (e.g. DMSO).
  • Figure 1 Clinical and radiographic findings in a 13 year old male with PPD (family 11).
  • a. left hand Note enlargement of the proximal interphalangeal joints (arrows)
  • b. hand radiograph demonstrating enlargement of the epiphyseal and metaphyseal portions of all hand bones. Arrows point to the same proximal interphalangeal joints as in (a)
  • c. knee radiograph demonstrating enlargement of the femoral and tibial epiphyses and a reduction in cartilage thickness indicated by the narrowing of the joint space (arrows)
  • Lateral spine radiograph demonstrating flattening and anterior beaking (arrow) of the thoraco-lumbar spine.
  • FIG. 1 Schematic depicting wisp3 protein domains and the mutations identified in patients with PPD. above. Sequencing gels demonstrating the compound heterozygous WISP 3 mutations found in family 3. Sequence is derived from subclones containing wild type or mutant patient-derived PCR amplimer. The direction of translation is indicated by the vertical arrow above the wild type codon. below. Locations of the likely disease causing WISP3 mutations. Families in which they were found are indicated within the circled numbers. All mutations were inherited. The sites of alteration relative to the putative 354 amino acid residue unprocessed polypeptide are indicated by the arrows. Numbering for the missense and nonsense mutations correspond to the amino acid residue.
  • Frameshift mutations (italics) correspond to the nucleotide residues.
  • the two homozygous frameshift mutations are underlined. Domains having homology to non-CCN protein families are indicated (IGF-BP, insulin like growth factor binding protein; VWC, Von Willebrand type C repeat; THBS, thrombospondin type 1 repeat; CK, cysteine knot).
  • IGF-BP insulin like growth factor binding protein
  • VWC Von Willebrand type C repeat
  • THBS thrombospondin type 1 repeat
  • CK cysteine knot
  • Figure 3 WISP 3 is expressed in skeletally-derived cells.
  • the ethidium bromide stained 3% agarose gel depicts products of amplification by RT-PCR from 5 different cell types for 4 genes.
  • PCR reactions using each primeitpair (COL11A2, WISP3, CACP-1, and UBE1) were performed separately on reverse transcribed total RNA and then pooled prior to electrophoresis. All primer pairs span large introns; only products derived from the spliced transcripts are present. Lanes: low molecular weight standard; negative control (RT-PCR using a water-only template);
  • EBV-transformed lymphoblast RNA template primary human fibroblast RNA template; primary human synoviocyte RNA template; primary human chondrocyte RNA template; bone-marrow-derived mesenchymal progenitor cell RNA from day 14 of chondrogenic differentiation.
  • COL11A2 encodes the a2 chain of type XI collagen, a quantitatively minor cartilage collagen. The gene is also transcribed in
  • CACP-1 encodes the protein defective in the Camptodactyly-Arthropathy-Coxa Vara-Pericarditis syndrome (manuscript submitted).
  • UBE1 encodes ubiqitin activating enzyme El, a putative "housekeeping" protein.
  • a partially complementary sequence is one that at least partially inhibits a completely complementary sequence from performing its function (e.g. enzymatic, binding, etc) in vivo or in vitro and is referred to using the functional term "substantially homologous.”
  • the inhibition function of the completely complementary sequence may be examined using an enzymatic assay, a binding assay or other assay designed to measure the particular function of the completely complementary protein.
  • the present invention contemplates WISP3 nucleic acid amplified from genomic DNA and mRNA, and substantially homologous sequences.
  • a "substantially homologous sequence” or probe will compete for and inhibit the function (e.g., the binding or enzymatic function) of a sequence which is completely homologous to a target under conditions of low stringency. This is not to say that conditions of low stringency are such that non-specific interaction is permitted; low stringency conditions require that the interaction of the sequence with its substrate be a specific (i.e., selective) interaction.
  • the absence of non-specific binding may be tested by the use of a second target which lacks even a partial degree of complementarity (e.g., less than about 30% identity); in the absence of non-specific interaction the probe will not react to the second non-complementary target.
  • a partial degree of complementarity e.g., less than about 30% identity
  • Low stringency conditions when used in reference to nucleic acid hybridization comprise conditions equivalent to binding or hybridization at 42°C in a solution consisting of 5X SSPE (43.8 g/1 NaCl, 6.9 g/1 NaH 2 PO 4 « H 2 O and 1.85 g/1 EDTA, pH adjusted to 7.4 with NaOH), 0.1% SDS, 5X Denhardt's reagent [50X Denhardt's contains per 500 ml: 5 g Ficoll (Type 400, Pharmacia), 5 g BSA (Fraction V; Sigma)] and 100 ⁇ g/ml denatured salmon sperm DNA followed by washing in a solution comprising 5X SSPE, 0.1% SDS at 42°C when a probe of about 500 nucleotides in length is employed.
  • 5X SSPE 43.8 g/1 NaCl, 6.9 g/1 NaH 2 PO 4 « H 2 O and 1.85 g/1 EDTA, pH adjusted to 7.4 with NaOH
  • 5X Denhardt's reagent
  • High stringency conditions when used in reference to nucleic acid hybridization comprise conditions equivalent to binding or hybridization at 42°C in a solution consisting of 5X SSPE (43.8 g/1 NaCl, 6.9 g/1 NaH 2 PO 4 « H 2 O and 1.85 g/1 EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS, 5X Denhardt's reagent and 100 ⁇ g/ml denatured salmon sperm DNA followed by washing in a solution comprising 0.1X SSPE, 1.0% SDS at 42°C when a probe of about 500 nucleotides in length is employed.
  • the art knows well that numerous equivalent conditions may be employed to comprise either low or high stringency conditions; factors such as the length and nature (DNA, RNA, base composition) of the probe and nature of the target (DNA, RNA, base composition, present in solution or immobilized, etc.) and the concentration of the salts and other components (e.g. , the presence or absence of formamide, dextran sulfate, polyethylene glycol) are considered and the hybridization solution may be varied to generate conditions of either low or high stringency hybridization different from, but equivalent to, the above listed conditions.
  • factors such as the length and nature (DNA, RNA, base composition) of the probe and nature of the target (DNA, RNA, base composition, present in solution or immobilized, etc.) and the concentration of the salts and other components (e.g. , the presence or absence of formamide, dextran sulfate, polyethylene glycol) are considered and the hybridization solution may be varied to generate conditions of either low or high stringency hybridization different from, but equivalent to, the above
  • Stringency when used in reference to nucleic acid hybridization typically occurs in a range from about T m -5°C (5°C below the T m of the probe) to about 20°C to 25 °C below T m .
  • a stringent hybridization can be used to identify or detect identical polynucleotide sequences or to identify or detect similar or related polynucleotide sequences.
  • stringent conditions a nucleic acid sequence of interest will hybridize to its exact complement and closely related sequences.
  • fusion protein refers to a chimeric protein containing the protein of interest (i.e., Wisp3 and fragments thereof) joined to an exogenous protein fragment (the fusion partner which consists of a non-Wisp3 sequence).
  • the fusion partner may provide a detectable moiety, may provide an affinity tag to allow purification of the recombinant fusion protein from the host cell, or both. If desired, the fusion protein may be removed from the protein of interest by a variety of enzymatic or chemical means known to the art.
  • purified or “to purify” refers to the removal of contaminants from a sample.
  • the present invention contemplates purified composi- tions (discussed above).
  • the term “partially purified” refers to the removal of a moderate portion of the contaminants of a sample to the extent that the substance of interest is recognizable by techniques known to those skilled in the art as accounting for a measurable amount of the mixture.
  • the present invention contemplates purifed, partially purified and/or substantially purified Wisp3 protein, and portions thereof for use as a lubricant.
  • substantially purified refers to the removal of a significant portion of the contaminants of a sample to the extent that the substance of interest is recognizable by techniques known to those skilled in the art as the most abundant substance in the mixture.
  • portion when in reference to a protein (as in “a portion of a given protein”) refers to fragments of that protein. The fragments may range in size from four amino acid residues to the entire amino acid sequence minus one amino acid.
  • the present invention contemplates "functional portions” of a protein. Such portions are “functional” if they contain a binding region
  • Such “functional portions” of the Wisp3 gene product are typically greater than 50 amino acids in length, and more typically greater than 100 amino acids in length. “Functional portions” may also be “conserved portions” of the protein. The present invention contemplates conserved portions 20 amino acids in length or greater, and more typically greater than 50 amino acids in length.
  • portion when in reference to an oligonucleotide sequence (as in “a portion of a given sequence”) refers to fragments of that sequence.
  • the fragments may range in size from four base residues to the entire oligonucleotide sequence minus one base. More typically, such portions are 15 nucleotides in length or greater. Again, such portions may be conserved portions. On the other hand, such portions may be unique portions of the gene.
  • “Staining” shall be defined as any number of processes known to those in the field that are used to better visualize, distinguish or identify a specific component(s) and/or feature(s) of a cell or cells.
  • “Morphology” shall be defined as the visual appearance of a cell or organism when viewed with the eye, a light microscope, a confocal microscope or an electron- microscope, as appropriate.
  • "In operable combination”, “in operable order” and “operably linked” as used herein refer to the linkage of nucleic acid sequences in such a manner that a nucleic acid molecule capable of directing the transcription of a given gene and/or the synthesis of a desired protein molecule is produced.
  • the present invention contemplates the WISP3 gene in operable combination with a promoter.
  • the term also refers to the linkage of amino acid sequences in such a manner so that a functional protein is produced.
  • Heterologous DNA refers to a nucleotide sequence which is not endogenous to the cell into which it is introduced.
  • Heterologous DNA includes a nucleotide sequence which is ligated to, or is manipulated to become ligated to, a nucleic acid sequence to which it is not ligated in nature, or to which it is ligated at a different location in nature.
  • Heterologous DNA also includes a nucleotide sequence which is naturally found in the cell into which it is introduced and which contains some modification relative to the naturally-occurring sequence.
  • An example of heterologous DNA of the present invention comprises the Wisp3 DNA sequence introduced into yeast.
  • “Expression vector” shall be defined as a sequence of DNA or RNA, in operable combination that is used to transfect a cell or cells. The sequence may be single or double stranded.
  • the present invention contemplates an expression vector comprising the WISP3 gene.
  • "Patient” shall be defined as a human or other animal, such as a guinea pig or mouse and the like, capable of having cell cycle (influenced) determined diseases, either naturally occurring or induced, including but not limited to cancer.
  • PPD is an autosomal recessive disorder that may be initially misdiagnosed as juvenile rheumatoid arthritis. Its population incidence has been estimated at 1 per million in the United Kingdom, but it is likely to be higher in the Middle East and Gulf States. Affected individuals are asymptomatic in infancy and in early childhood.
  • Symptoms begin in mid-childhood and consist of stiffness and swelling of joints, motor weakness, and joint contractures (Fig 1). Symptoms often are first noticed in the hands, then in the knees, hips, spine and other large joints as the disease progresses. In older children and in adults, radiographic examination distinguishes PPD from rheumatoid arthritis, by showing loss of joint space, widened epiphyses, and vertebral flattening (Fig 1). Interestingly, radiographic findings are not apparent in infants and young children. Skeletal radiographs, taken when 3 years of age, were available for two unrelated children who subsequently developed symptoms of PPD; in neither child did the these studies reveal an underlying skeletal dysplasia.
  • Cartilage appears to be the primary affected tissue, and in one patient, a biopsy of the iliac crest revealed abnormal nests of chondrocytes and loss of normal cell columnar organization in growth zones.
  • the present invention contemplates preparations comprising synovium lubricants (e.g. Wisp3 protein, or portions thereof).
  • synovium lubricants e.g. Wisp3 protein, or portions thereof.
  • Said Wisp3 protein may be purified from source tissue (e.g. bovine sources) or produced using recombinant technology (see, generally, Sambrook et al. Molecular Cloning: A Laboratory Manual, 2d ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., and Current Protocols in Molecular Biology (1996) John Wiley and Sons, Inc., N.Y., which are incorporated herein by reference) which is taught throughout this document. All the information contained therein is incorporated herein by reference.
  • Such formulations can be prepared either as liquid solutions or suspensions, or in solid forms.
  • Formulations may include such normally employed additives such as binders, fillers, carriers, preservatives, stabilizing agents, emulsifiers, buffers and excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations, or powders, and typically contain l%-95% of active ingredient, preferably 2%-70%.
  • additives such as binders, fillers, carriers, preservatives, stabilizing agents, emulsifiers, buffers and excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like.
  • These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations, or powders, and typically contain l%-95%
  • compositions are also prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared.
  • injectables either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared.
  • the present invention contemplates formulations comprising one or more synovium lubricants along with one or more local anesthetic. It is not intended that the present invention be limited to particular anesthetics. A variety are contemplated including but not limited to procaine or lidocaine. When injecting a bursa, tendon sheath, or periarticular region, such a mixture will give immediate relief (due to the anesthetic) followed by more lasting relief (due to the lubricant).
  • a topical anesthetic prior to injection may be used.
  • topical anethetics include but are not limited to ethyl chloride spray on the skin over the joint to be injected.
  • a local anesthetic may be given first, followed by administration of one or more of the above- described lubricants.
  • synovium lubricants can be given orally, applied as creams or ointments or injected (including but not limited to intravenous injection and intra- 5 articular injection).
  • the present invention specifically contemplates intra-articular injections in patients.
  • the specific area of the joint to be injected is palpated and is then marked, e.g. , with firm pressure by a ballpoint pen that has the inked portion retracted. This will leave an impression that will last 10 to 30 minutes.
  • the ballpoint pen technique can also be used with soft tissue injection.
  • the area to be aspirated and/or injected should be carefully cleansed with a good antiseptic, such as one of the iodinated compounds. Then the needle can be inserted through the ballpoint pen impression.
  • Helpful equipment includes the following items: alcohol sponges; iodinated
  • the knee is the easiest joint to inject.
  • the patient should be in a supine position with the knee fully extended.
  • the puncture mark is made just posterior to the medial portion of the patella, and an 18- to 20-gauge, 1 1/2-inch needle directed slightly posteriorly and slightly inferiorly.
  • the joint space should be entered readily. On occasion thickened synovium or villous projections may occlude the opening of the
  • infrapatellar plica a vestigal structure that is also called the ligamentum mucosum, may prevent adequate aspiration of the knee when the medial approach is used.
  • the plica should not adversely affect injections or aspirations from the lateral aspect. Shoulder. Injections in the shoulder are most easily accomplished with the patient sitting and the shoulder externally rotated. A mark is made just medial to the head of the humerus and slightly inferiorly and laterally to the coracoid process. A 20- to 22-gauge, 1 1/2-inch needle is directed posteriorly and slightly superiorly and laterally. One should be able to feel the needle enter the joint space. If bone is hit, the operator should pull back and redirect the needle at a slightly different angle.
  • the acromioclavicular joint may be palpated as a groove-at the lateral end of the clavicle just medial to the shoulder. A mark is made, and a 22- to 25-gauge, 5/8- to 1-inch needle is carefully directed inferiorly. Rarely is synovial fluid obtained.
  • the sternoclavicular joint is most easily entered from a point directly anterior to the joint. Caution is necessary to avoid a pneumotharax.
  • the space is fibrocartilaginous, and rarely can fluid be aspirated.
  • Ankle Joint For injections of the lubricants of the present invention in the ankle joints, the patient should be supine and the leg-foot angle at 90 degrees. A mark is made just medical to the tibialis anterior tendon and lateral to the medial malleolus. A 20- to 22-gauge, 1 1/2-inch needle is directed posteriorly and should enter the joint space easily without striking bone.
  • Subtalar Ankle Joint Again, the patient is supine and the leg-foot angle at 90 degrees. A mark is made just inferior to the tip of the lateral malleolus. A 20- to 22- gauge, 1 1/2-inch needle is directed perpendicular to the mark. With this joint the needle may not enter the first time, and another attempt or two may be necessary.
  • Wrist This is a complex joint, but notably most of the intercarpal spaces communicate.
  • a mark is made just distal to the radius and just ulnar to the so-called anatomic snuff box.
  • anatomic snuff box usually a 24- to 26-gauge, 5/8 to 1-inch needle is adequate, and the injection is made perpendicular to the mark. If bone is hit, the needle should be pulled back and slightly redirected toward the thumb.
  • First Carpometacarpal Joint Degenerative arthritis often involves this joint. Frequently the joint space is quite narrowed, and injections may be difficult and painful. A few simple maneuvers may make the injection fairly easy, however. The thumb is flexed across the palm toward the tip of the fifth finger. A mark is made at the base of the first metacarpal bone away from the border of the snuff box. A 22- to 26-gauge, 5/8 to 1-inch needle is inserted at the mark and directed toward the proximal end of the fourth metacarpal. This approach avoids hitting the radial artery. Metacarpophalalangeal Joints and Finger Interphalangral Joints.
  • Synovitis in these joints usually causes the synovium to bulge dorsally, and a 24- to 26-gauge, 1/2 to 5/8-inch needle can be inserted on the either side just under the extensor tendon mechanism. It is not necessary for the needle to be interposed between the articular surfaces. Some prefer having the fingers slightly flexed when injecting the metacarpophalangeal joints. It is unusual to obtain synovial fluid. When injecting, a mix of the lubricants of the present invention with a small amount of local anesthetic is preferred.
  • Metatarsophalangeal Joints and Toe Interphalangeal Joints are quite similar to those of the metacapophalangeal and finger interphalangeal joints, but many prefer to inject more dorsally and laterally to the extensor tendons. Marking the area(s) to be injected is helpful as is gentle traction on the toe of each joint that is injected.
  • Elbow A technique preferred by many is to have the elbow flexed at 90 degrees.
  • the joint capsule will bulge if there is inflammation.
  • a mark is made just below the lateral epicondyle of the humerus.
  • a 22-gauge, 1 to 1 1/2-inch is inserted at the mark and directed parallel to the shaft of the radius or directed perpendicular to the skin.
  • Hip This is a very difficult joint to inject even when, using a fluoroscope as a guide. Rarely is the physician quite sure that the joint has been entered; synovial fluid is rarely obtained.
  • Two approaches can be used, anterior or lateral. A 20-gauge, 3 1/2-inch spinal needle should be used for both approaches.
  • the patient is supine and the extremity fully extended and externally rotated.
  • a mark should be made about 2 to 3 cm below the anterior superior iliac spine and 2 to 3 cm lateral to the femoral pulse.
  • the needle is inserted at a 60 degree angle to the skin and directed posteriorly and medially until bone is hit.
  • the needle is withdrawn slightly, and possibly a drop or two of synovial fluid can be obtained, indicating entry into the joint space.
  • the needle can "follow" the femoral neck into the joint.
  • the patient is supine, and the hips should be internally rotated - the knees apart and toes touching.
  • a mark is made just anteriorlo the greater trochanter, and the needle is inserted and directed medially and sightly cephalad toward a point slightly below the middle of the inguinal ligament. One may feel the tip of the needle slide into the joint.
  • Temporomandibular Joint For injections, the tempormandibular joint is palpated as a depression just below the zygomatic arch and 1 to 2 cm anterior to the tragus. The depression is more easily palpated by having the patient open and close the mouth.
  • a mark is made and, with the patient's mouth open, a 22-gauge, 1/2 to 1- inch needle is inserted perpendicular to the skin and directed slightly posteriorly and superiorly.
  • the present invention may be used as an experimental control in assays used for the screening of compounds that may act as therapeutics in the treatment of osteoarthritis.
  • the present invention may be used as a known standard in in vitro and in vivo assays known to those practiced in the art.
  • the present invention contemplates detecting Wisp3 protein.
  • the present invention contemplates obtaining Wisp3 protein from patients (e.g. from joint tissue or fluid) in order to detect and or measure Wisp3 protein.
  • Antibodies to Wisp3 can be conveniently used to monitor Wisp3 and Wisp3 levels. Such assays can be done in liquid or solid phase.
  • the present invention contemplates the use of antibodies to Wisp3 in an ELISA (or similar) format.
  • antibodies to Wisp3 can be used in Western blot assays.
  • Wisp3 protein levels are measured to follow the progression of disease. In another embodiment, Wisp3 protein levels are measured to detect the response to treatment.
  • WISP 3 BLAST the non-redundant and EST databases. Two potential candidates were identified. One was LAMA4, the other was an EST (IMAGE clone 1076664) which has homology to the CCN gene family. This EST is now known to contain part of WISP 3.
  • LAMA4 LAMA4
  • EST IMAGE clone 1076664
  • This EST is now known to contain part of WISP 3.
  • WISP3 cDNA sequence Pieris, D. et. al. "WISP genes are members of the connective tissue growth factor family that are up-regulated in Wnt-1 -transformed cells and aberrantly expressed in human colon tumors" Proc. Natl Acad. Sci. 95:14717-14722, 1998) to the genomic sequence from the PAC. Exon 1 is not contained within the PAC sequence. Amplification and Sequencing of WISP3 in families with PPD and in unaffected controls
  • cDNA was prepared from total RNA using the superscript preamplif ⁇ cation system (GibcoBRL). 25 ⁇ L PCR reactions contained 2 ⁇ L template cDNA, 0.2 ⁇ M each primer, and 200 ⁇ M each dNTP.
  • PPD was mapped to a 3 cM interval on chromosome 6q22 by performing linkage studies in consanguineous families (El-Shanti, H., et al. "Assignment of ⁇ ene responsible for progressive pseudorheumatoid dysplasia to chromosome 6 and examination of COL10A1 as candidate gene" Eur. J. Hum. Genet. 6:251-256, 1998; Fischer, J. et. al. "Genetic linkage of progressive pseudorheumatoid dysplasia to a 3-cM interval of chromosome 6q22" Hum. Genet. 103:60-64, 1998).
  • D6S1706 (data not shown).
  • the fully linked marker D6S416 is contained within this interval.
  • D6S416 is also present in the completely sequenced PAC dJ142L7 (GenBank accession # Z99289). This PAC sequence contains exons for two genes, LAMA4 and WISP3.
  • WISP3 is a member of the CCN family of putative growth regulators (Pennica, D. et. al. "WISP genes are members of the connective tissue growth factor family that are up-regulated in Wnt-1 -transformed cells and aberrantly expressed in human colon tumors" Proc. Natl Acad. Sci. 95:14717-14722, 1998).
  • cysteines are essential to each family member's function (Bork, P. "The modular architecture of a new family of growth regulators related to connective tissue growth factor" FEBS Lett. 327:125-130,
  • WISP genes are members of the connective tissue growth factor family that are up-regulated in Wnt-1 -transformed cells and aberrantly expressed in human colon tumors" Proc. Natl. Acad. Sci. 95:14717-14722, 1998). Little is known about the expression pattern and function o WISP3 (Pennica, D. et. al "WISP genes are members of the connective tissue growth factor family that are up-regulated in
  • WISP genes are members of the connective tissue growth factor family that are up-regulated in Wnt-1 -transformed cells and aberrantly expressed in human colon tumors" Proc. Natl Acad. Sci. 95:14717-14722, 1998; O'Brien, T.P., et al. "Expression of cyr ⁇ l, a growth factor-inducible immediate-early gene" Mol. Cell Biol 10:3569-3577, 1990; Ryseck, R.-P., et al.
  • the human analog of murine cystein rich protein 16 is a l(,25-dihydroyvitamin D 3 responsive immediate early gene in human fetal osteoblasts: regulation by cytokines, growth factors, and serum" Endocrinology 139:1761-1770, 1998); one family member Cyr ⁇ l is expressed at sites of mesenchymal cell differentiation into chondrocytes (O'Brien, T.P., et al. "Expression of cyr ⁇ l, a growth factor-inducible immediate-early gene" Mol. Cell Biol. 10:3569-3577, 1990; Wong, M., et al.
  • CCN family members have a modular architecture with putative functional domains roughly corresponding to each exon (Bork, P. "The modular architecture of a new family of growth regulators related to connective tissue growth factor" FEBS Lett.
  • the peptide signal sequence is encoded by exon 1.
  • Exon 2 encodes an N-terminal domain that has homology to the insulin-like growth factor binding proteins (IGF-BP).
  • Exon 3 encodes a domain with homology to Von Willebrand factor type C (VWC) repeats and may participate in peptide oligomerization; however, WISP3, unlike all other CCN family members identified to date, lacks 4 of 10 conserved cysteine residues in this domain (Pennica, D. et. al. "WISP genes are members of the connective tissue growth factor family that are up-regulated in Wnt-1 -transformed cells and aberrantly expressed in human colon tumors" Proc. Natl. Acad. Sci.
  • Exon 4 encodes a thrombospondin type I domain and may be involved in the binding of CCNs to sulfated glycosaminoglycans either at cell surfaces or in extracellular matrix.
  • Exon 5 encodes a "cysteine knot" domain, that has been identified in several other signaling peptides (e.g. transforming growth factor b, platelet derived growth factor, and nerve growth factor), and may participate in dimerization and receptor binding.
  • signaling peptides e.g. transforming growth factor b, platelet derived growth factor, and nerve growth factor
  • WISP genes are members of the connective tissue growth factor family that are up-regulated in Wnt-1 -transformed cells and aberrantly expressed in human colon tumors" Proc-. Natl. Acad. Sci. 95:14717-14722, 1998; Zhang, R. et al. "Identification of rCop-1, a new member of the ccn protein family, as a negative regulator for cell transformation" Mol. Cell Biol. 18:6131-6141, 1998).
  • WISP3 has two closely related homologues, F75Pi and WISP2, which were identified through their increased expression in Wnt-1 transformed cells (Pennica, D. et. al. "WISP genes are members of the connective tissue growth factor family that are up-regulated in Wnt-1 -transformed cells and aberrantly expressed in human colon tumors" Proc. Natl. Acad. Sci. 95:14717-14722, 1998).
  • the Wnt family of signaling proteins also regulate cell fate, motility, morphology, and proliferation (Dale, T.C. "Signal transduction by the Wnt family of ligands" Biochem. J.
  • WISP1 and WISP2 demonstrate differential expression in primary human colon cancers.
  • WISP3 was identified by database searching (Pennica, D. et. al. "WISP genes are members of the connective tissue growth factor family that are up-regulated in Wnt-1 -transformed cells and aberrantly expressed in human colon tumors" Proc.
  • WISP3 is differentially expressed in primary human colon tumors, relative to its expression in the patient's normal mucosa (Pennica, D. et. al "WISP genes are members of the connective tissue growth factor family that are up-regulated in Wnt-1 -transformed cells and aberrantly expressed in human colon tumors" Proc. Natl. Acad. Sci. 95:14717-14722, 1998), the physiological relevance of this observation is unknown. The lack of extraskeletal manifestations in patients with PPD suggests that WISP3's primary, non-redundant role is in the regulation of post-natal skeletal growth and long term skeletal homeostasis.
  • WISP3 genomic DNA Although we can easily detect WISP3 genomic DNA by Southern blot, we have been unable to detect transcripts with commercially available murine northern blots and multi-tissue adult human northern blots, even after long exposures. In addition, few WISP3 sequences are contained within the human and murine EST databases. This suggests WISP3 is likely to have a low and tissue-specific pattern of expression. Interestingly, expression of another CCN family member has also been difficult to detect (Zhang, R. et al. "Identification of rCop-1, a new member of the ccn protein family, as a negative regulator for cell transformation" Mol. Cell Biol. 18:6131-6141, 1998).
  • WISP 3 cDNA has been amplified from several tissues including kidney, testis, placenta, ovary, prostate, and small intestine (Pennica, D. et. al. "WISP genes are members of the connective tissue growth factor family that are up-regulated in Wnt-1 -transformed cells and aberrantly expressed in human colon tumors" Proc. Natl. Acad. Sci. 95:14717-14722, 1998).
  • the present invention provides reagents and methods for the screening of compounds that can be used as therapeutics for Osteoarthritis, as well as providing reagents and methods for the treatment of Osteoarthritis.

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Abstract

La présente invention concerne des procédés et compositions se rapportant au traitement de l'arthrose. Ce traitement fait intervenir une nouvelle classe de médicaments contre l'arthrose, en l'occurrence des composés convenant comme lubrifiants du tissu où l'arthrose a été diagnostiquée. L'invention concerne également des réactifs de recherche systématique de composés convenant comme agents thérapeutiques pour le traitement de l'arthrose.
PCT/US2000/018938 1999-07-23 2000-07-11 Nouveaux procedes et reactifs convenant au traitement de l'arthrose WO2001007085A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002033085A2 (fr) * 2000-10-16 2002-04-25 Genentech, Inc. Techniques de traitement utilisant des polypeptides wisp
WO2005025603A2 (fr) * 2003-09-11 2005-03-24 Genentech, Inc. Procedes d'utilisation d'antagonistes de wisp

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998021236A1 (fr) * 1996-11-08 1998-05-22 Human Genome Sciences, Inc. Facteur de croissance 3 du tissu conjonctif

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998021236A1 (fr) * 1996-11-08 1998-05-22 Human Genome Sciences, Inc. Facteur de croissance 3 du tissu conjonctif

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002033085A2 (fr) * 2000-10-16 2002-04-25 Genentech, Inc. Techniques de traitement utilisant des polypeptides wisp
WO2002033085A3 (fr) * 2000-10-16 2003-04-17 Genentech Inc Techniques de traitement utilisant des polypeptides wisp
US7687460B2 (en) 2000-10-16 2010-03-30 Genentech, Inc, Methods of treatment using wisp polypeptides
US8138152B2 (en) 2000-10-16 2012-03-20 Genentech, Inc. Methods of treatment using WISP polypeptides
WO2005025603A2 (fr) * 2003-09-11 2005-03-24 Genentech, Inc. Procedes d'utilisation d'antagonistes de wisp
WO2005025603A3 (fr) * 2003-09-11 2006-04-27 Genentech Inc Procedes d'utilisation d'antagonistes de wisp

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