US20030069177A1 - Method for treating cartilage disorders - Google Patents

Method for treating cartilage disorders Download PDF

Info

Publication number
US20030069177A1
US20030069177A1 US09/858,935 US85893501A US2003069177A1 US 20030069177 A1 US20030069177 A1 US 20030069177A1 US 85893501 A US85893501 A US 85893501A US 2003069177 A1 US2003069177 A1 US 2003069177A1
Authority
US
United States
Prior art keywords
igf
seq
igfbp
peptide
binding
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.)
Abandoned
Application number
US09/858,935
Other languages
English (en)
Inventor
Yves Dubaquie
Ellen Filvaroff
Henry Lowman
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.)
Genentech Inc
Genetech Inc
Original Assignee
Genentech Inc
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Genentech Inc filed Critical Genentech Inc
Priority to US09/858,935 priority Critical patent/US20030069177A1/en
Assigned to GENETECH, INC. reassignment GENETECH, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: DUBAQUIE, YVES, LOWMAN, HENRY B., FILVAROFF, ELLEN
Priority to US10/271,869 priority patent/US7423017B2/en
Publication of US20030069177A1 publication Critical patent/US20030069177A1/en
Priority to US11/929,468 priority patent/US7947650B2/en
Priority to US11/934,582 priority patent/US8110548B2/en
Abandoned legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • 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
    • 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
    • A61K38/22—Hormones
    • A61K38/30—Insulin-like growth factors, i.e. somatomedins, e.g. IGF-1, IGF-2
    • 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
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/04—Drugs for skeletal disorders for non-specific disorders of the connective tissue
    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4743—Insulin-like growth factor binding protein
    • 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
    • C07K14/575—Hormones
    • C07K14/65—Insulin-like growth factors, i.e. somatomedins, e.g. IGF-1, IGF-2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates generally to the treatment of cartilage disorders, including stimulation of cartilage repair and treatment of degenerative cartilagenous disorders.
  • Degenerative cartilagenous disorders broadly describe a collection of diseases characterized by degeneration or metabolic abnormalities of the connective tissues that are manifested by pain, stiffness and limitation of motion of the affected body parts. The origin of these disorders can be pathological or as a result of trauma or injury.
  • Osteoarthritis also known as osteoarthrosis or degenerative joint disease
  • OA osteoarthritis
  • the incidence of OA increases with age, and evidence of OA involvement can be detected in some joints in the majority of the population by age 65.
  • OA is often also accompanied by a local inflammatory component that may accelerate joint destruction.
  • OA is characterized by disruption of the smooth articulating surface of cartilage, followed by formation of clefts and fibrillation, and ultimately by the full-thickness loss of the cartilage.
  • alterations of the periarticular bone include the development of palpable bone enlargements at the joint margins and deformity resulting from assymetric cartilage destruction.
  • OA symptoms include local pain at the affected joints, especially after use. With disease progression, symptoms may develop into a continuous aching sensation, local discomfort, and cosmetic alterations of the affected joint.
  • rheumatoid arthritis is a systematic destructive and debilitating disease that is believed to begin in the synovium, the tissues surrounding the joint.
  • the prevalance of RA is about 1 ⁇ 6 that of OA in the general population of the United States. It is a chronic autoimmune disorder characterized by symmetrical synovitis of the joint and typically affects small and large diarthrodial joints, leading to their progressive destruction.
  • the symptoms of RA may also include fever, weight loss, thinning of the skin, multi-organ involvement, scleritis, corneal ulcers, the formation of subcutaneous or subperiosteal nodules, and premature death.
  • cartilage is avascular and mature chondrocytes have little intrinsic potential for replication, mature cartilage has limited ability for repair. Thus, damage to the cartilage layer that does not penetrate to the subchondral bone does not undergo efficient repair. In contrast, when the subchondral bone is penetrated, its vascular supply allows a triphasic repair to take place. The resulting tissue is usually mechanically sub-optimal fibrocartilage.
  • the degradation associated with osteoarthritis usually initially appears as fraying and fibrillation of the surface. Loss of proteoglycan from the matrix also occurs. As the surface fibrillation progresses, the defects penetrate deeper into the cartilage, resulting in loss of cartilage cells and matrix. The subchondral bone thickens, is slowly exposed, and may appear polished. Bony nodules or osteophytes also often form at the periphery of the cartilage surface and occasionally grow over the adjacent eroded areas. If the surface of these bony outgrowths is permeated, vascular outgrowth may occur and cause the formation of tissue plugs containing fibrocartilage.
  • IL-1 ⁇ interleukin-1-alpha
  • NO nitric oxide
  • the cytokine IL-1 ⁇ has catabolic effects on cartilage, including the generation of synovial inflammation and up-regulation of matrix metalloproteinases and prostaglandin expression (Baragi et al., J. Clin. Invest., 96: 2454-2460 (1995); Baragi et al., Osteoarthritis Cartilage, 5: 275-282 (1997); Evans et al., J. Keukoc. Biol., 64: 55-61 (1998); Evans and Robbins, J.
  • IL-1 ⁇ interleukin-1-alpha
  • NO nitric oxide
  • IL-1ra soluble IL-1 receptor antagonist
  • Cartilage obtained from osteoarthritic joints endogenously produces large amounts of NO.
  • Normal cartilage does not produce NO unless stimulated with cytokines such as IL-1, while osteoarthritic cartilage explants continue to express NO synthase for up to 3 days in culture despite the absence of added stimuli.
  • cytokines such as IL-1
  • osteoarthritic cartilage explants continue to express NO synthase for up to 3 days in culture despite the absence of added stimuli.
  • the inhibition of NO has been shown to prevent IL-1 ⁇ -mediated cartilage destruction and chondrocyte death as well as the progression of osteoarthritis.
  • peptide growth factors are very significant regulators of cartilage growth and cell behavior (i.e., differentiation, migration, division, or matrix synthesis and/or breakdown) (Chen et al., Am J. Orthop., 26: 396-406 (1997)). These factors are under investigation for their potential to induce host cartilage repair without transplantation of cells, and are being incorporated into engineered devices for implantation.
  • growth factors are soluble proteins of relatively small molecular mass that are rapidly absorbed and/or degraded, a great challenge exists in making them available to cells in sufficient quantity and for sufficient duration. It is likely desirable to have different factors present at the repair site during different parts of the developmental cycle, and for varying lengths of time.
  • the ideal delivery vehicle is biocompatible and resorbable, has the appropriate mechanical properties, and results in no harmful degradation products.
  • Growth factors that previously have been proposed to stimulate cartilage repair include insulin-like growth factor-I (IGF-1) (Osborn, J. Orthop. Res., 7: 35-42 (1989); Florini and Roberts, J. Gerontol., 35: 23-30 (1980); U.S. Pat. No.
  • bFGF basic fibroblast growth factor
  • BMP bone morphogenetic protein
  • TGF- ⁇ transforming growth factor beta
  • IGF-1 has been administered with sodium pentosan polysulfate (PPS) (a chondrocyte catabolic activity inhibitor) to severely osteoarthritic canines with the effect of reducing the severity of the disease perhaps by lowering the levels of active neutral metalloproteinase in the cartilage.
  • PPS pentosan polysulfate
  • IGF-1 and PPS together appeared to successfully maintain cartilage structure and biochemistry, while IGF alone was ineffective, as described in Rogachefsky, Osteoarthritis and Cartilage, 1: 105-114 (1993); Rogachefsky et al., Ann. NY Acad. Sci., 732: 889-895 (1994).
  • the use of IGF-1 either alone or as an adjuvant with other growth factors to stimulate cartilage regeneration has been described in WO 91/19510, WO 92/13565, U.S. Pat. No. 5,444,047, and EP 434,652.
  • IGF-1 has also been found useful in the treatment of osteoporosis in mammals exhibiting decreased bone mineral density and those exposed to drugs or environmental conditions that result in bone density reduction and potentially osteoporosis, as described in EP 560,723 and EP 436,469.
  • IGF-1 insufficiency may have an etiologic role in the development of osteoarthritis (Coutts et al., “Effect of growth factors on cartilage repair,” Instructional Course Lect., 47: 487-494 (Amer. Acad. Orthop. Surg.: Rosemont, Ill. 1997)).
  • Some studies indicate that serum IGF-1 concentrations are lower in osteoarthritc patients than control groups, while other studies have found no difference. Nevertheless, it has been shown that both serum IGF-1 levels and chrondrocyte responsiveness to IGF-1 decrease with age, with the latter likely due to high levels of IGF binding proteins (IGFBPs) (Florini and Roberts, J.
  • IGFBPs IGF binding proteins
  • IGFBP-3 appears to be the most responsible for regulating the total levels of IGF-1 and IGF-2 in plasma.
  • IGFBP-3 is a GH-dependent protein and is reduced in cases of GH-deficiency or resistance (Jones et al., supra; Rosenfield et al., “IGF-1 treatment of syndromes of growth hormone insensitivity” In: The insulin - like growth factors and their regulatory proteins , Eds Baxter R C, Gluckman P D, Rosenfield R G. Excerpta Medica, Amsterdam, 1994), pp 357-464; Scharf et al., J. Hepatology, 25: 689-699 (1996)).
  • IGFBPs are able to enhance or inhibit IGF activity, depending largely on their post-translational modifications and tissue localization (reviewed in Jones and Clemmons, Endocr. Rev. 16:3-34 (1995); Collet-Solberg and Cohen, Endocrinol. Metabol. Clin. North Am. 25:591-614 (1996)).
  • disregulation in IGFBPs may play a key role in arthritic disorders (Chevalier and Tyler, Brit. J. Rheum. 35: 515-522 (1996); Olney et al., J. Clin. Endocrinol. Metab. 81: 1096-1103 (1996); Martel-Pelletier et al., Inflamm.
  • IGF-1 analogs with very low binding affinity for IGFBPs were more effective than wild-type IGF-1 in stimulating proteoglycan synthesis (Morales, Arch Biochem. Biophys. 324, 173-188 (1997)). More recent data, however, suggest that IGFBPs contribute to IGF binding to and transport through cartilage tissue, and IGFBPs may thus regulate bioavailability of IGF-1 within the joint (Bhakta et al., J. Biol. Chem., 275: 5860-5866 (2000)).
  • IGF-1 insulin growth factor-1
  • IGFBP-3 IGFBP-3
  • ALS acid-labile subunit
  • This ternary complex of 150-kD molecular weight is unable to traverse the vasculature walls and acts as a circulating reservoir for IGF's.
  • the serum half-life of IGF-1 in ternary complexes is reported to be 12-15 hours, as opposed to 30 minutes in binary complexes, or 10 minutes in the free form (Simpson et al., Growth Horm IGF Res, 8: 83-95 (1998); Twigg and Baxter, J. Biol. Chem., 273: 6074-6079 (1998)).
  • IGFBP-3 and -5 are apparently unique in their ability to form a ternary complex with ALS. ALS association occurs only in the presence of IGF-1, and a basic motif in the carboxy-terminal domains of IGFBP-3 and -5 seems to mediate this interaction (Baxter et al., J. Biol. Chem., 267: 60-65 (1992); Firth et al., J. Biol. Chem., 273: 2631-2638 (1998); Twigg and Baxter, supra).
  • IGFBP-3 is the most abundant binding protein, followed by IGFBP-1 and -2 levels, whereas the serum concentrations of IGFBP-4, -5, and -6 are quite low (Clemmons, Cytokine Growth Factor Rev., 8: 45-62 (1997)). IGFBP-3 therefore represents the main IGF-1 carrier in the blood. In contrast, a substantial portion of IGFBP-1 and -2 in the blood are unoccupied. Hence, they appear to be the major modulators of free IGF-1 levels (Clemmons, 1997, supra).
  • WO 94/04569 discloses a specific binding molecule, other than a natural IGFBP, that is capable of binding to IGF-1 and can enhance the biological activity of IGF-1.
  • WO 98/45427 published Oct. 15, 1998; Lowman et al., Biochemistry, 37: 8870-8878 (1998); and Dubaquie and Lowman, Biochemistry, 38: 6386 (1999) disclose IGF-1 agonists identified by phage display.
  • WO 97/39032 discloses ligand inhibitors of IGFBP's and methods for their use. Further, U.S. Pat. No.
  • 5,891,722 discloses antibodies having binding affinity for free IGFBP-1 and devices and methods for detecting free IGFBP-1 and a rupture in a fetal membrane based on the presence of amniotic fluid in a vaginal secretion, as indicated by the presence of free IGFBP-1 in the vaginal secretion.
  • WO 00/23469 published Apr. 27, 2000 discloses fragments of IGFBPs and analogs of IGF-1 for use in, e.g., cancer, ischemic injury, and diabetes treatment.
  • the present invention concerns a method of treating a cartilage disorder as claimed, comprising contacting cartilage with an effective amount of an active agent selected from an IGF-1 analog with a binding affinity preference for IGFBP-3 over IGFBP-1, an IGF-1 analog with a binding affinity preference for IGFBP-1 over IGFBP-3, or an IGFBP displacer peptide that prevents the interaction of IGF with IGFBP-3 or IGFBP-1 and does not bind to a human IGF receptor.
  • an active agent selected from an IGF-1 analog with a binding affinity preference for IGFBP-3 over IGFBP-1, an IGF-1 analog with a binding affinity preference for IGFBP-1 over IGFBP-3, or an IGFBP displacer peptide that prevents the interaction of IGF with IGFBP-3 or IGFBP-1 and does not bind to a human IGF receptor.
  • the cartilage is treated in vivo in a mammal and the active agent is administered to the mammal.
  • the active agent is optionally contacted with the cartilage in an extended-release form and/or administered locally to the joint alone or, if the active agent is an IGFBP displacer peptide or IGF-1 analog with a preference for IGFBP-3 over IGFBP-1, together with IGF-1 and/or ALS, preferably human, native-sequence IGF-1 if the mammal is human.
  • the active agent is an IGF-1 variant wherein the amino acid residue at position 3, 7, 10, 16, 25, or 49, or the amino acid residues at positions 3 and 49 of native-sequence human IGF-1 are replaced with an alanine, a glycine, or a serine residue, or an IGF-1 variant wherein the amino acid residue at position 9, 12, 15, or 20 is replaced with a lysine or arginine residue, or an IGFBP-3 displacer peptide designated as: Y24LY31A IGF-1; 4D3.3P; BP3-4D3.11; BP3-4D3.11DEL; BP3-4B3.3; BP3-01-ox; BP3-02-ox; BP3-06; BP3-08; BP3-15; BP3-16; BP3-17; BP3-25; BP3-27; BP3-28; BP3-30; BP3-39; BP3-40; BP3-41; BP3-107; or BP3
  • the letter followed by a number followed by a letter indicates an IGF-1 analog wherein the amino acid letter to the left of the number is the original amino acid in native-sequence human IGF-1, the number is the position where the amino acid is changed, and the amino acid letter to the right of the number is the substituted amino acid.
  • F49A indicates an IGF-1 variant wherein the phenylalanine residue at position 49 of native-sequence human IGF-1 is changed to an alanine residue
  • E3AF49A indicates an IGF-1 variant wherein the glutamine residue at position 3 of native-sequence human IGF-1 is changed to an alanine residue, and the phenylalanine residue at position 49 of native-sequence human IGF-1 is changed to an alanine residue.
  • the above method is for the treatment of cartilage damaged or diseased as a result of a degenerative cartilagenous disorder.
  • the disorder is an articular cartilage disorder, and most preferably is OA or RA.
  • the above method is for the treatment of joints damaged directly or indirectly by injury, preferably microdamage or blunt trauma, a chondral fracture, an osteochondral fracture.
  • the invention concerns the above treatment method wherein the cartilage is contacted with an effective amount of the IGF-1 analog or IGFBP displacer peptide as defined above in combination with an effective amount of a cartilage growth factor or cartilage catabolism antagonist.
  • the invention concerns a method of maintaining, enhancing, or promoting the growth of chondrocytes in serum-free culture by contacting the chondrocytes with an effective amount of an IGF-1 analog or an IGFBP displacer peptide as identified above.
  • the method concerns contacting the chondrocyte with an effective amount of an IGF-1 analog or an IGFBP displacer peptide in an extended-release formulation.
  • the present invention concerns a method of stimulating the regeneration or preventing the degradation of cartilage resulting from injury or a degenerative cartilagenous disorder by transplantation of an effective amount of chondrocytes previously treated with an effective amount of an IGF-1 analog or an IGFBP displacer peptide as defined above.
  • the present invention concerns an article of manufacture comprising a container holding an IGF-1 analog or an IGFBP displacer peptide as defined above in a pharmaceutically acceptable carrier with instructions for its use in treating a cartilage disorder.
  • FIGS. 1 A- 1 C depict the DNA sequence (SEQ ID NO:1) of plasmid pt4.g8 used as a template to construct a phage library. Also shown is the amino acid sequence (SEQ ID NO:2) of an antibody-recognizable (gD-tag) peptide fused to g8p of bacteriophage M13.
  • FIG. 2 shows gene-8 naive phage library enrichments with a selection using four library pools each and the targets IGF-1, IGFBP-L, and IGFBP-3.
  • FIG. 3 shows an IGF-1 blocking assay using g8-phage peptides from IGFBP-3 selections, where the phage titration is with 100 nM IGF-1.
  • the open circles are peptide 4A3.1
  • the open triangles are peptide 4B3.4
  • the open squares are peptide 4C3.2
  • the solid circles are peptide 4D3.3
  • the solid triangles are peptide 4D3.4
  • the solid squares are peptide 4D3.5.
  • FIG. 4 shows an IGF-1 blocking assay using g8-phage peptides from IGFBP-3 selections, where the phage titration is without IGF-1.
  • the designations for the peptides are the same as those described above for FIG. 3.
  • FIG. 5 shows an IGF-1 blocking assay using g8-phage peptides from IGFBP-3 selections, where the peptides (4C3.2, 4D3.8, 4D3.9, 4D3.11, and 4D3.12) are from a NEUTRAVIDINTM/DTT selection.
  • the solid bars are with 100 ⁇ M IGF-1 and the open bars are without IGF-1.
  • FIG. 6 shows an IGF-1 blocking assay using g8-phage peptides from IGFBP-3 selections where the peptides (indicated on the x axis) are from direct-coat/HCl selection.
  • the solid bars are with 100 ⁇ M IGF-1 and the open bars are without IGF-1.
  • FIG. 7 depicts a competition assay of IGFBP-3 inhibition by a peptide binding to IGFBP-3 (designated BP3-01) using a BIACORETM surface-plasmon-resonance device to measure free binding protein.
  • the circles indicate 800 response units (RU) of IGF-1 and the squares indicate 400 RU of immobilized IGF-1.
  • FIG. 8 depicts a competition assay of IGFBP-3 inhibition by a peptide binding to IGFBP-3 (designated BP3-02) using a BIACORETM surface-plasmon-resonance device to measure free binding protein.
  • the circles indicate 800 RU of IGF-1 and the squares indicate 400 RU of immobilized IGF-1.
  • FIG. 9 shows a radiolabeled IGF-1 plate assay of the ability of two peptides that bind to IGFBP-3 but not to the Type 1 IGF receptor (BP3-01-ox: circles, and BP3-02-ox: squares) to inhibit IGFBP-3.
  • FIG. 10 shows a radiolabeled IGF-1 plate assay of the ability of the two IGFBP-3 binding peptides described for FIG. 9 to inhibit IGFBP-1 (symbols are the same).
  • FIGS. 11 A- 11 D depict KIRA assays of IGF-1 activity using three peptides (BP1-01: squares, BP1-02: circles, and BP03-ox: triangles).
  • FIG. 11A depicts the peptides alone
  • FIG. 11B depicts the peptides plus IGF-1 plus IGFBP-1
  • FIG. 11C depicts the peptides plus IGF-1
  • FIG. 11D depicts the peptides plus IGF-1 plus IGFBP-3.
  • FIG. 12 depicts an IGF-2 competition assay of IGFBP-3 inhibition by four peptides, designated BP3-01-ox (open squares), BP3-14 (open circles), BP3-15 (closed circles), and BP3-17 (closed squares), using a BIACORETM surface-plasmon-resonance device to measure free binding protein. Each peptide was tested using 20 nM IGFBP-3 and approximately 1500 RU of immobilized IGF-2.
  • FIGS. 13A and 13B show a phage ELISA of the variant, G1S-A70V IGF-1, binding to IGFBP-1 (FIG. 13A) and IGFBP-3 (FIG. 13B).
  • Microtiter plates coated with 1 ⁇ g/ml IGFBP-1 (FIG. 13A) or IGFBP-3 (FIG. 13B) were incubated with phage particles displaying G1S-A70V in the presence of the indicated amounts of soluble competitor protein, IGFBP-1 (FIG. 13A) or IGFBP-3 (FIG. 13B).
  • the half-maximal inhibitory concentration (IC 50 ) of competitor i.e., the inhibitory concentration of competitor that resulted in half-maximal binding of the phagemid in that particular experiment, is denoted for the respective IGFBP.
  • FIG. 14 shows the loss or gain of IGFBP affinity for the IGF-1 mutants tested by phage ELISA.
  • Relative IC 50 values IC 50mut /IC 50 G1S-A70V ) of each IGF-1 alanine mutant (affinity changes of each mutant for the binding proteins with respect to IGF-1 G1S-A70V) are shown for IGFBP-1 (filled bars) and IGFBP-3 (open bars). Data are taken from Table I below.
  • Relative IC 50 values ⁇ 1 denote gain of affinity; values >1 denote loss of affinity. The asterisk indicates that these particular variants were not displayed on phage, as judged by antibody binding.
  • FIGS. 15A and 15B show binding specificity of the IGF-1 variant F49A displayed on phage to IGFBP-1 and -3, respectively, in competitive-phage ELISA.
  • Phagemid particles displaying F49A squares
  • IGFBP-3 IGFBP-3
  • Immunosorbent plates were coated with 1 ⁇ g/ml IGFBP-3 and ELISA were carried out as described in the Examples below using wild-type IGF-1 phage (WT, circles) and IGF-F49A phage (F49A, squares) in parallel. Experiments were carried out in duplicate, and data points are shown as mean ⁇ standard deviation.
  • FIG. 16 discloses a sequence alignment of native-sequence human IGF-1 (designated wtIGF)(SEQ ID NO:3), native-sequence human proinsulin (designated proinsulin) (SEQ ID NO:4), and native-sequence human insulin (designated insulin (B chain) followed by insulin (A chain)) (SEQ ID NO:5).
  • the asterisks and dots indicate sequence identity and sequence similarity, respectively, at the indicated amino acid positions among the three sequences.
  • FIGS. 17 A- 17 D show a biosensor analysis of IGFBP binding to immobilized IGF-1 variants. Sensorgrams are shown for IGFBP-1 (FIGS. 17A, 17C) or IGFBP-3 (FIGS. 17B, 17D) binding to immobilized wild-type IGF-1 (FIGS. 17A, 17B) or F49A IGF variant (FIGS. 17C, 17D).
  • concentrations of ligand in each experiment were 1 ⁇ M, 500 nM, and 250 nM. See Table II for kinetic parameters.
  • FIGS. 18 A- 18 B show a model of the functional binding epitopes for IGFBP-1 and IGFBP-3, respectively, on the surface of IGF-1.
  • Amino acid side chains were classified according to their relative contribution in binding energy (Table I) and colored as follows: no effect (grey); 2-5 fold loss of apparent affinity (yellow); 5-10 fold (orange); 10-100 fold (bright red); >100 fold (dark red). If available, numbers from phage ELISA experiments in Table I below were used.
  • BIACORETTM data were used instead for V11A, R36A, and P39A variants (Table II).
  • the NMR structure of IGF-1 (Cooke et al., Biochemistry, 30: 5484, (1991)) was represented using the program Insight IITM (MSI, San Diego, Calif.).
  • the binding epitope for IGFBP-1 (FIG. 18A) is located on the “upper” and “lower” face of the N-terminal helix (residues 8-17), connected by the energetically-important residue F49.
  • IGFBP-3 FIG. 18B
  • individual IGF-1 side chains contribute very little binding energy.
  • the binding epitope has shifted away from the N-terminus and newly includes G22, F23, Y24.
  • FIG. 19 shows the amount of bound IGFBP-1, determined in a competitive BIACORETM binding experiment, plotted against the IGF variant concentration for E3A/F49A (squares) and F49A (circles).
  • FIGS. 20A and 20B show, respectively, the calculated IGF-1 activity in nM units for several IGF-1 variants at 13 nM (high) and 1.3 nM (low) variant concentrations using IGF-1 KIRA optical density analysis.
  • the signal obtained for each IGF variant was compared to that of a standard-dilution series of wild-type IGF-1, and reported in terms of an apparent IGF-1 concentration corresponding to the observed activity.
  • FIGS. 21A and 21B show IGF receptor activation curves for F49A IGF-1 (FIG. 21A) and E3A/F49A (FIG. 21B) as well as for wild-type IGF-1, as measured using serial dilutions in KIRA assays.
  • the variants are represented by squares and the wild-type IGF-1 is represented by circles.
  • FIGS. 22A and 22B show an assessment of preliminary pharmacological properties of F49A and E3A/F49A IGF-1, radiolabeled and administered intravenously to rats.
  • FIG. 22A shows a time course of the rate at which both molecules are cleared from the blood of the animals, where the squares represent wild-type IGF-1, the circles represent E3A/F49A IGF-1, and the diamonds represent F49A IGF-1.
  • FIG. 22A shows a time course of the rate at which both molecules are cleared from the blood of the animals, where the squares represent wild-type IGF-1, the circles represent E3A/F49A IGF-1, and the diamonds represent F49A IGF-1.
  • FIG. 22B shows the tissue-to-blood ratio for these two IGF variants in different organs, namely, kidney, liver, spleen, heart, and pancreas, at 5, 15, and 30 minutes, where the solid bars represent wild-type IGF-1, the dotted bars represent E3A/F49A IGF-1, and the striped bars represent F49A IGF-1.
  • FIG. 23 shows circular dichroism spectra of wild-type IGF-1 (circles), F49A IGF-1 (squares), and E3A/F49A IGF-1 (diamonds).
  • FIG. 24 is a bar graph showing the effect of control, wild-type IGF-1, F49A, and E3A/F49A (at a concentration of 40 or 400 ng/ml) on cartilage matrix breakdown (proteoglycan release at 72 hours).
  • FIG. 25 is a bar graph showing the effect of wild-type IGF-1, F49A, and E3A/F49A (at a concentration of 40 or 400 ng/ml) on IL1 ⁇ -induced cartilage breakdown at 72 hours.
  • FIG. 26 is a bar graph showing the effect of control, wild-type IGF-1, F49A, E3A/F49A (at a concentration of 40 or 400 ng/ml) on matrix synthesis.
  • FIG. 27 is a bar graph showing the effect of wild-type IGF-1, F49A, and E3A/F49A (at a concentration of 40 or 400 ng/ml) on IL1 ⁇ -induced inhibition of matrix synthesis.
  • FIG. 28 is a bar graph showing the effect of control, wild-type IGF-1, F49A and E3A/F49A (at a concentration of 40 or 400 ng/ml) on nitric oxide release.
  • FIG. 29 is a bar graph showing the effect of wild-type IGF-1, F49A, and E3A/F49A (at a concentration of 40 or 400 ng/ml) on IL1 ⁇ -induced nitric oxide production.
  • FIGS. 30A and 30B show the binding curves for phage particles displaying either wild-type IGF-1 (circles), D12K (squares), or D12R (diamonds) bound to immobilized IGFBP-1 (FIG. 30A) or IGFBP-3 (FIG. 30B).
  • FIGS. 31 A- 31 D show the effects on porcine articular cartilage explants cultured in media ( ⁇ ) or media with D12K, D12R, or wild-type IGF-1 (at 10 nM) alone (FIGS. 31A, 31C) or in the presence of IL-1 ⁇ (+a) at 1 ng/ml (FIGS. 31B, 31D).
  • FIG. 32 shows the effect on articular cartilage matrix synthesis in human tissue from diseased joints cultured in media alone ( ⁇ ) or with F49, E3A/F49, F16/F49, D12K, D12R or wild-type IGF-1 (at 40 ng/ml).
  • FIGS. 33 A- 33 D show the effect on human articular cartilage explants cultured in media ( ⁇ ) or treated with wild-type IGF-1 by itself or in combination with either BP3-40 (FIGS. 33A, 33B) or BP3-15 (FIGS. 33C, 33D) (at 0.1 mg/ml).
  • FIGS. 34 A- 34 C show the trimeric complex formation of F49A or E3A/F49A with IGFBP-3 and ALS.
  • IGFBP-3 immobilized on a biosensor chip was saturated by including 1 ⁇ M wild-type IGF-1 (FIG. 34A), F49A (FIG. 34B), or E3A/F49A (FIG. 34C) in the running buffer.
  • ALS was injected at 98 nM, 148 nM, and 33 nM, monitoring real-time association and dissociation to the preformed binary complex.
  • FIG. 35 shows a BIAcoreTM inhibition assay of IGF-I activity using seven different peptides (BP1-16: filled circles, (i+7)A: open circles, (i+7)B: open diamonds, (i+7)C: open triangles, (i+7)D: open squares, (i+8)B: filled squares, (i+8)C: filled triangles).
  • FIG. 36 shows a KIRA assay of peptide activity using four different peptides (BP1-16: circles, BP1-02: squares, BP1-25: triangles, and BP1-40: diamonds).
  • FIG. 37 shows an analytical HPLC run of the trypsin-cleaved BP1-625-Z fusion. The major peaks were identified by mass spectrometry as (A) Z-domain fragment and (B) BP1-625 peptide.
  • FIG. 38 shows a BIAcoreTM inhibition assay of IGF-I activity using four different peptides (BP1-01: circles, BP1-625: squares, BP1-21A: triangles, and BP1-25: diamonds).
  • FIG. 39 shows the effect on proteoglycan synthesis of articular cartilage explants from human joints removed from patients undergoing joint replacement cultured with IGF-1 alone (IGF) at 40 ng/ml, or IGF-1 with BP1-17, BP3-15, or BP1-16 (0.1 mg/ml), or IGF-1 with buffer (HEPES).
  • IGF IGF-1 alone
  • HEPES IGF-1 with buffer
  • IGF-1 analogs are amino acid variants of native-sequence IGF-1, preferably variants of human wild-type IGF-1.
  • the dissociation constant (K D ) of wild-type IGF-1 was determined to be 13 nM for IGFBP-1 and 1.5 nM for IGFBP-3.
  • the difference in affinity for the IGFBP's is due to a 10-fold faster association rate (k a ) of IGF-1 to IGFBP-3 (3.2 ⁇ 10 5 versus 3.2 ⁇ 10 4 M ⁇ 1 s ⁇ 1 ).
  • Such analogs may have one or more amino acid alterations as compared to native IGF-1.
  • IGF-1 analogs refers either to an IGF-1 analog with a binding affinity preference for IGFBP-3 over IGFBP-1 or an IGF-1 analog with a binding affinity preference for IGFBP-1 over IGFBP-3, as defined below.
  • “Peptides” have at least two amino acids and include polypeptides having at least about 50 amino acids.
  • the definition includes peptide derivatives, their salts, or optical isomers.
  • the IGFBP displacer peptide is an IGFBP-3 or IGFBP-1 displacer peptide.
  • a peptide that “binds to IGFBP-3” or “binds to IGFBP-l” refers to a peptide that binds IGFBP-3 or IGFBP-1 to at least some degree, whether with high affinity or not.
  • human IGF receptor refers to any receptor for an IGF found in humans and includes the Type 1 and Type 2 IGF receptors in humans to which both human IGF-1 and IGF-2 bind, such as the placental Type 1 IGF-1 receptor, etc.
  • a peptide that “does not bind to a human IGF receptor” does not bind at all to any such receptor, or binds to such receptor with an affinity more than about 200-fold less than wild-type human IGF-1 (hIGF-1) or wild-type human IGF-2 (hIGF-2) binds to such receptor.
  • the peptide binds to such receptor with an affinity of more than about 250-fold less than wild-type hIGF-1 or hIGF-2 binds to the same receptor or does not bind at all.
  • cartilage disorder refers to any injury or damage to cartilage, and to a collection of diseases that are manifested by symptoms of pain, stiffness, and/or limitation of motion of the affected body parts. Included within the scope of “cartilage disorders” is “degenerative cartilagenous disorders”, which is a colllection of disorders characterized, at least in part, by degeneration or metabolic derangement of connective tissues of the body, including not only the joints or related structures, including muscles, bursae (synovial membrane), tendons, and fibrous tissue, but also the growth plate, meniscal system, and intervertebral discs.
  • the term “degenerative cartilagenous disorders” includes “articular cartilage disorders,” which are characterized by disruption of the smooth articular cartilage surface and degradation of the cartilage matrix. Additional pathologies include nitric oxide production, and inhibition or reduction of matrix synthesis. Included within the scope of “articular cartilage disorder” are OA and RA. Examples of degenerative cartilagenous disorders include systemic lupus erythematosus and gout, amyloidosis or Felty's syndrome.
  • the term covers the cartilage degradation and destruction associated with psoriatic arthritis, kidney disorders, osteoarthrosis, acute inflammation (e.g., yersinia arthritis, pyrophosphate arthritis, gout arthritis (arthritis urica), and septic arthritis), arthritis associated with trauma, ulcerative colitis (e.g., Crohn's disease), multiple sclerosis, diabetes (e.g., insulin-dependent and non-insulin dependent), obesity, giant cell arthritis, and Sjogren's syndrome.
  • the disorder is microdamage or blunt trauma, a chondral fracture, or an osteochondral fracture.
  • OA osteoarthritis
  • OA defines not a single disorder, but the final common pathway of joint destruction resulting from multiple processes. OA is characterized by localized assymetric destruction of the cartilage commensurate with palpable bone enlargements at the joint margins. OA typically affects the interphalangeal joints of the hands, the first carpometacarpal joint, the hips, the knees, the spine, and some joints in the midfoot, while large joints, such as the ankles, elbows, and shoulders, tend to be spared.
  • OA can be associated with metabolic diseases such as hemochromatosis and alkaptonuria, developmental abnormalities such as developmental dysplasia of the hips (congenital dislocation of the hips), limb-length descrepancies, including trauma and inflammatory arthritides such as gout, septic arthritis, and neuropathic arthritis. OA may also develop after extended mechanical instability, such as resulting from sports injury or obesity.
  • metabolic diseases such as hemochromatosis and alkaptonuria
  • developmental abnormalities such as developmental dysplasia of the hips (congenital dislocation of the hips)
  • limb-length descrepancies including trauma and inflammatory arthritides such as gout, septic arthritis, and neuropathic arthritis.
  • trauma and inflammatory arthritides such as gout, septic arthritis, and neuropathic arthritis.
  • OA may also develop after extended mechanical instability, such as resulting from sports injury or obesity.
  • RA rheumatoid arthritis
  • RA is a systemic, chronic, autoimmune disorder characterized by symmetrical synovitis of the joint and typically affects small and large diarthroid joints alike.
  • symptoms may include fever, weight loss, thinning of the skin, multiorgan involvement, scleritis, corneal ulcers, the formation of subcutaneous or subperiosteal nodules, and even premature death.
  • the symptoms of RA often appear during youth and can include vasculitis, atrophy of the skin and muscle, subcutaneous nodules, lymphadenopathy, splenomegaly, leukopaenia, and chronic anaemia.
  • Treatment is an intervention performed with the intention of preventing the development or altering the pathology of a disorder.
  • treatment refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted pathological condition or disorder.
  • Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented.
  • a therapeutic agent may directly decrease or increase the magnitude of response of a pathological component of the disorder, or render the disease more susceptible to treatment by other therapeutic agents, e.g., antibiotics, antifungals, anti-inflammatory agents, chemotherapeutics, etc.
  • treatment includes a method for the prevention of initial or continued damage or disease of joints by degenerative cartilagenous disorders and/or injury.
  • the term “effective amount” is the minimum efficacious concentration of the IGF analog or IGFBP displacer peptide as set forth herein. This includes the minimum concentration of such protein or peptide that causes, induces, or results in either a detectable improvement or repair of damaged cartilage or a measurable protection from continued or induced cartilage destruction, such as the inhibition of synthesis or loss of proteoglycans from cartilage tissue.
  • Cartilage growth factor refers to agent(s) other than an IGF-1 analog or an IGFBP displacer peptide as identified herein that cause, induce, or result in an improvement in the condition of or protection from initial or continued destruction of cartilage subject to damage by either injury or a degenerative cartilagenous disorder.
  • Such cartilage growth factors include insulin-like growth factors (e.g., IGF-1, IGF-2), platelet-derived growth factors (PDGFs), bone morphogenic proteins (BMPs), transforming growth factor- ⁇ s (1-3), members of the epidermal growth factor family (e.g., EGF, HB-EGF, TGF- ⁇ ), and fibroblast growth factors (FGFs).
  • Cartilage catabolism antagonists are those agents that inhibit, attenuate or otherwise block the activity or effect of molecules that are associated with or aggravate cartilage destruction.
  • IL-1 ⁇ and nitric oxide (NO) are agents known to be associated with cartilage destruction.
  • IL1ra direct (IL1ra) or indirect (IL-4 or IL-10) inhibitors of IL-1 ⁇ or other inflammatory cytokines (e.g., TNF- ⁇ ) and NO production
  • cartilage catabolism antagonists include antagonists of chondrocyte catabolism (e.g., sodium pentosan polysulfate, glucosamine (and variants thereof, such as mannosamine) or chondroitin sulfate, tetracycline, hyaluronan)
  • agents that inhibit catabolism of cartilage indirectly, for example through their effects on the underlying, subchondral bone e.g., bisphosphonates or osteoprotegerin (OPG)
  • Chronic administration refers to administration of the agent(s) in a continuous mode as opposed to an acute mode, so as to maintain the initial therapeutic effect (activity) for an extended period of time.
  • Intermittent administration is treatment that is not consecutive without interruption, but rather is cyclic in nature.
  • mammal for purposes of treatment refers to any animal classified as a mammal, including humans, domestic, and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, sheep, pigs, cows, etc.
  • the preferred mammal herein is a human.
  • non-adult refers to mammals that are from perinatal age (such as low-birth-weight infants) up to the age of puberty, the latter being those that have not yet reached full growth potential.
  • Administration “in combination with” one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order.
  • Carriers as used herein include pharmaceutically-acceptable carriers, excipients, or stabilizers that are non-toxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Often the physiologically-acceptable carrier is an aqueous pH-buffered solution.
  • physiologically-acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low-molecular-weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counter-ions such as sodium; hyaluronan; and/or non-ionic surfactants such as TWEENTM, polyethylene glycol (PEG), and PLURONICSTM.
  • buffers such as phosphate, citrate, and other organic acids
  • antioxidants including ascorbic acid
  • a “liposome” is a small vesicle composed of various types of lipids, phospholipids, and/or surfactants that is useful for delivery of a drug (such as the IGF-1 analog or IGFBP displacer peptide disclosed herein) to a mammal.
  • a drug such as the IGF-1 analog or IGFBP displacer peptide disclosed herein.
  • the components of the liposome are commonly arranged in a bilayer formation, similar to the lipid arrangement of biological membranes.
  • extended-release or “sustained-release” formulations in the broadest possible sense means a formulation of active IGF-1 analog or IGFBP displacer peptide identified herein resulting in the release or activation of the active analog or peptide for a sustained or extended period of time—or at least for a period of time that is longer than if the analog or peptide were made available in vivo in the native or unformulated state.
  • the extended-release formulation occurs at a constant rate and/or results in sustained and/or continuous concentration of the active agent herein.
  • Suitable extended-release formulations may comprise microencapsulation, semi-permeable matrices of solid hydrophobic polymers, biogradable polymers, biodegradable hydrogels, suspensions, or emulsions (e.g., oil-in-water or water-in-oil).
  • the extended-release formulation comprises poly-lactic-co-glycolic acid (PLGA) and can be prepared as described in Lewis, “Controlled Release of Bioactive Agents form Lactide/Glycolide polymer,” in Biodegradable Polymers as Drug Delivery Systems, M. Chasin and R. Langeer, Ed. (Marcel Dekker, New York), pp. 1-41.
  • the extended-release formulation is stable and the activity of the IGF-1 analog or IGFBP displacer peptide as identified herein does not appreciably diminish with storage over time. More specifically, such stability can be enhanced through the presence of a stabilizing agent such as a water-soluble polyvalent metal salt.
  • a stabilizing agent such as a water-soluble polyvalent metal salt.
  • IGF-1 refers to insulin-like growth factor-1 from any species, including bovine, ovine, porcine, equine, and human, preferably human, and, if referring to exogenous administration, from any source, whether natural, synthetic, or recombinant.
  • “Native-sequence” human IGF-1 the sequence of which is shown in FIG. 16 (SEQ ID NO:3), is prepared, e.g., by the process described in EP 230,869 published Aug. 5, 1987; EP 128,733 published Dec. 19, 1984; or EP 288,451 published Oct. 26, 1988. More preferably, this native-sequence IGF-1 is recombinantly produced.
  • IGF-2 refers to insulin-like growth factor-2 from any species, including bovine, ovine, porcine, equine, and human, preferably human, and, if referring to exogenous administration, from any source, whether natural, synthetic, or recombinant. It may be prepared by the method described in, e.g., EP 128,733.
  • IGEBP or an “IGF binding protein” refers to a protein or polypeptide normally associated with or bound or complexed to IGF-1 or IGF-2, whether or not it is circulatory (i.e., in serum or tissue). Such binding proteins do not include receptors.
  • This definition includes IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-5, IGFBP-6, Mac 25 (IGFBP-7), and prostacyclin-stimulating factor (PSF) or endothelial cell-specific molecule (ESM-1), as well as other proteins with high homology to IGFBPs.
  • Mac 25 is described, for example, in Swisshelm et al., Proc. Natl. Acad. Sci.
  • ALS acid-labile subunit
  • the invention herein relates to the use of an IGF-1 analog or an IGFBP displacer peptide as defined above to treat cartilage disorders, preferably degenerative cartilagenous disorders, including regenerating and/or preventing the degradation of cartilage.
  • IGF-1 analogs with a binding affinity preference for IGFBP-3 over IGFBP-1 include an IGF-1 variant wherein the amino acid(s) of wild-type human IGF-1 at position 3, 7, 10, 16, 25, or 49 or at positions 3 and 49 of native-sequence human IGF-1 are replaced with an alanine, a glycine, and/or a serine residue.
  • an alanine or glycine residue Preferably, one or both of the amino acids in question are substituted by an alanine or glycine residue, most preferably alanine.
  • the more preferred IGF-1 analog with such binding affinity preference herein is F49A, F49G, F49S, E3A, E3G, E3S, E3AF49A, E3AF49G, E3AF49S, E3GF49A, E3GF49G, E3GF49S, E3SF49A, E3SF49G, E3SF49S, F16A, F16G, F16S, F16AF49A, F16GF49A, F16SF49A, F16SF49A, F16AF49S, F16AF49G, F16SF49S, F16SF49G, F16GF49S, or F16GF49G.
  • IGF-1 analogs with a binding affinity preference for IGFBP-1 over IGFBP-3 include an IGF-1 variant wherein the amino acid(s) of wild-type human IGF-1 at position 9, 12, 15, or 20 is/are replaced with a lysine or arginine residue.
  • the more preferred IGF-1 analog with such binding affinity preference herein is D12K or D12R.
  • IGFBP-3 displacer peptides include a peptide selected from the group consisting of:
  • BP3-4D3.11 VAWEVCWDRHDQGYICTTDS (SEQ ID NO:7);
  • BP3-4D3.11DEL (AWEVCWDRHQGYICTTDS) (SEQ ID NO:8);
  • BP3-4B3.3 (EESECFEGPGYVICGLVG) (SEQ ID NO:9);
  • BP3-02-ox (DMGVCADGPWMYVCEWTE) (SEQ ID NO:11);
  • BP3-06 (TGVDCQC*GPVHC*VCMDWA)(SEQ ID NO:12);
  • BP3-08 (TVANCDC*YMPLC*LCYDSD) (SEQ ID NO:13);
  • BP3-15 SEEVCWPVAEWYLCN (SEQ ID NO:14);
  • BP3-16 (VCWPVAEWYLCNMWG) (SEQ ID NO:15);
  • BP3-17 (VCWPVAEWYLCN) (SEQ ID NO:16);
  • BP3-25 (CWPVAEWYLCN) (SEQ ID NO:17);
  • BP3-27 ECWPVAEWYLCN (SEQ ID NO:18);
  • BP3-28 (EEVCWPVAEWYLCN) (SEQ ID NO:19);
  • BP3-30 (ASEEVCWPVAEWYLCN) (SEQ ID NO:20);
  • BP3-39 SEEVCWPVAEWYLCN-nh2 (SEQ ID NO:21);
  • BP3-41 (GPETCWPVAEWYLCN) (SEQ ID NO:21);
  • BP3-108 (suc-IPVSPDWFVCQ-nh2) (SEQ ID NO:25);
  • the C* indicates a cysteine that has been linked to another cysteine in the peptide.
  • the remaining Cys pairs are also oxidized as disulfides in each peptide.
  • the more preferred IGFBP-3 displacer peptide herein is BP3-15, BP3-39, BP3-40, BP3-01-OX, BP3-27, BP3-28, BP3-30, BP3-41, or 4D3.3P.
  • the most preferred IGFBP-3 displacer peptide herein is BP3-15, BP3-39, or BP3-40.
  • IGFBP-1 displacer peptides include a peptide selected from the group consisting of:
  • BP1-04 (CRAGPLQWLCE) (SEQ ID NO:28);
  • BP1-10 (CRKGPLQWLCELYF) (SEQ ID NO:29);
  • BP1-11 (CRKGPLQWLCEKYF) (SEQ ID NO:30);
  • BP1-13 (CKEGPLLWLCEKYF) (SEQ ID NO:32);
  • BP1-14 SEVGCRAGPLQWLCEKYFG-nh2 (SEQ ID NO:33);
  • BP1-15 CAAGPLQWLCEKYF (SEQ ID NO:34);
  • BP1-18 (CRAGPLQWLCEKAA) (SEQ ID NO:37);
  • BP1-19 SEMVCRAGPLQWLCEIYF-nh2* (SEQ ID NO:38);
  • BP1-20 (EARVCRAGPLQWLCEKYF-nh2) (SEQ ID NO:39);
  • BP1-21A SEVGCRAGPLQWLCEKYFSTY-nh2 (SEQ ID NO:40);
  • BP1-21B (CRAGPLQWLCEKYFSTY-nh2) (SEQ ID NO:41);
  • BP1-25 (EARVCRAGPLQWLCEKYFSTY) (SEQ ID NO:42);
  • BP1-40 GQQSCRAGPLQWLCEKYFSTY (SEQ ID NO:43);
  • BP68 (CRAGPLQWLCEKFF) (SEQ ID NO:45);
  • BP1027 (CKAGPLLWLCERFF) (SEQ ID NO:48);
  • BP1028 (CRAGPLQWLCERFF) (SEQ ID NO:49);
  • BP1029 (CREGPLQWLCERFF) (SEQ ID NO:50);
  • the C* indicates a cysteine that has been linked to another cysteine in the peptide, and the remaining Cys pairs are also oxidized as disulfides in each peptide.
  • the more preferred IGFBP-1 displacer peptide herein is BP1-16, BP1-20, BP1-21A, BP1-25, BP1-40, BP625, BP625-Z, and BP625T; and most preferred are BP1-20, BP1-21A, BP1-25, BP1-40, BP1-625, BP1-625-Z, and BP1-625T.
  • the still more preferred active agents herein are F49A, E3A, F16A, E3AF49A, F16AF49A, D12K, D12R, BP3-15, BP3-40, BP3-39, BP1-16, BP1-20, BP1-21A, BP1-25, BP1-40, BP1-625, and BP1-625-Z; and the most preferred are F49A, E3AF49A, F16AF49A, D12K, D12R, BP3-15, BP3-40, BP3-39, BP1-20, BP1-21A, BP1-25, BP1-40, BP1-625, BP1-625-Z, and BP1-625T.
  • the IGF-1 analogs and IGFBP displacer peptides useful in accordance with this invention can be made by any means that are known in the art, including chemical synthesis or recombinant production. Chemical synthesis, especially solid phase synthesis, is preferred for short (e.g., less than 50 residues) peptides or those containing unnatural or unusual amino acids such as D-Tyr, Ornithine, amino adipic acid, and the like. Recombinant procedures are preferred for longer polypeptides. When recombinant procedures are selected, a synthetic gene may be constructed de novo or a natural gene may be mutated by, for example, cassette mutagenesis. Set forth below are exemplary general recombinant procedures.
  • a variation on the above procedures contemplates the use of gene fusions, wherein the gene encoding the desired analog or peptide is associated, in the vector, with a gene encoding another protein or a fragment of another protein.
  • the “other” protein or peptide is often a protein or peptide that can be secreted by the cell, making it possible to isolate and purify the desired analog or peptide from the culture medium and eliminating the necessity of destroying the host cells that arises when the desired analog or peptide remains inside the cell.
  • the fusion protein can be expressed intracellularly. It is useful to use fusion proteins that are highly expressed.
  • proteolytic cleavage of fusion protein (Carter, in Protein Purification: From Molecular Mechanisms to Large - Scale Processes , Ladisch et al., eds. (American Chemical Society Symposium Series No. 427, 1990), Ch 13, pages 181-193).
  • Proteases such as Factor Xa, thrombin, and subtilisin or its mutants, and a number of others have been successfully used to cleave fusion proteins.
  • a peptide linker that is amenable to cleavage by the protease used is inserted between the “other” protein (e.g., the Z domain of protein A) and the desired analog or peptide.
  • the nucleotide base pairs encoding the linker are inserted between the genes or gene fragments coding for the other proteins.
  • Proteolytic cleavage of the partially purified fusion protein containing the correct linker can then be carried out on either the native fusion protein, or the reduced or denatured fusion protein.
  • the analog or peptide may or may not be properly folded when expressed as a fusion protein. Also, the specific peptide linker containing the cleavage site may or may not be accessible to the protease. These factors determine whether the fusion protein must be denatured and refolded, and if so, whether these procedures are employed before or after cleavage.
  • An ⁇ -amino protecting group (a) must render the ⁇ -amino function inert under the conditions employed in the coupling reaction, (b) must be readily removable after the coupling reaction under conditions that will not remove side-chain protecting groups and will not alter the structure of the analog/peptide fragment, and (c) must eliminate the possibility of racemization upon activation immediately prior to coupling.
  • protecting groups known to be useful for analog/peptide synthesis will vary in reactivity with the agents employed for their removal.
  • certain protecting groups such as triphenylmethyl and 2-(p-biphenylyl)isopropyloxycarbonyl are very labile and can be cleaved under mild acid conditions.
  • protecting groups such as t-butyloxycarbonyl (BOC), t-amyloxycarbonyl, adamantyl-oxycarbonyl, and p-methoxybenzyloxycarbonyl are less labile and require moderately strong acids, such as trifluoroacetic, hydrochloric, or boron trifluoride in acetic acid, for their removal.
  • Still other protecting groups such as benzyloxycarbonyl (CBZ or Z), halobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl cycloalkyloxycarbonyl, and isopropyloxycarbonyl, are even less labile and require stronger acids, such as hydrogen fluoride, hydrogen bromide, or boron trifluoroacetate in trifluoroacetic acid, for their removal.
  • acids such as hydrogen fluoride, hydrogen bromide, or boron trifluoroacetate in trifluoroacetic acid
  • protection may be, for example, by C1-C4 alkyl, such as t-butyl; benzyl (BZL); substituted BZL, such as p-methoxybenzyl, p-nitrobenzyl, p-chlorobenzyl, o-chlorobenzyl, and 2,6-dichlorobenzyl.
  • C1-C4 alkyl such as t-butyl
  • BZL benzyl
  • substituted BZL such as p-methoxybenzyl, p-nitrobenzyl, p-chlorobenzyl, o-chlorobenzyl, and 2,6-dichlorobenzyl.
  • protection may be, for example, by esterification using groups such as BZL, t-butyl, cyclohexyl, cyclopentyl, and the like.
  • a protecting group such as tetrahydropyranyl, tert-butyl, trityl, BZL, chlorobenzyl, 4-bromobenzyl, or 2,6-dichlorobenzyl is suitably employed.
  • the preferred protecting group is 2,6-dichlorobenzyl.
  • xanthyl (Xan) is preferably employed.
  • the amino acid is preferably left unprotected.
  • Each protected amino acid or amino acid sequence is introduced into the solid-phase reactor in excess, and the coupling is suitably carried out in a medium of dimethylformamide (DMF) or CH 2 Cl 2 or mixtures thereof. If incomplete coupling occurs, the coupling procedure is repeated before removal of the N-amino protecting group prior to the coupling of the next amino acid.
  • the success of the coupling reaction at each stage of the synthesis may be monitored. A preferred method of monitoring the synthesis is by the ninhydrin reaction, as described by Kaiser et al., Anal. Biochem, 34: 595 (1970).
  • the coupling reactions can be performed automatically using well known methods, for example, a BIOSEARCH 9500TM peptide synthesizer.
  • the protected analog/peptide Upon completion of the desired analog/peptide sequence, the protected analog/peptide must be cleaved from the resin support, and all protecting groups must be removed. The cleavage reaction and removal of the protecting groups is suitably accomplished simultaneously or stepwise.
  • the bond anchoring the analog/peptide to the resin is an ester linkage formed between the free carboxyl group of the C-terminal residue and one of the many chloromethyl groups present on the resin matrix. It will be appreciated that the anchoring bond can be cleaved by reagents that are known to be capable of breaking an ester linkage and of penetrating the resin matrix.
  • One especially convenient method is by treatment with liquid anhydrous hydrogen fluoride.
  • This reagent not only will cleave the analog/peptide from the resin but also will remove all protecting groups. Hence, use of this reagent will directly afford the fully deprotected analog/peptide.
  • hydrogen fluoride treatment results in the formation of the free peptide acids.
  • benzhydrylamine resin is used, hydrogen fluoride treatment results directly in the free peptide amines. Reaction with hydrogen fluoride in the presence of anisole and dimethylsulfide at 0° C. for one hour will simultaneously remove the side-chain protecting groups and release the analog/peptide from the resin.
  • the protected analog/peptide-resin can undergo methanolysis to yield the protected analog/peptide in which the C-terminal carboxyl group is methylated.
  • the methyl ester is then hydrolyzed under mild alkaline conditions to give the free C-terminal carboxyl group.
  • the protecting groups on the analog/peptide chain then are removed by treatment with a strong acid, such as liquid hydrogen fluoride.
  • a strong acid such as liquid hydrogen fluoride.
  • a particularly useful technique for methanolysis is that of Moore et al., Peptides, Proc. Fifth Amer. Pept. Symp ., M. Goodman and J. Meienhofer, Eds., (John Wiley, N.Y., 1977), p. 518-521, in which the protected analog/peptide-resin is treated with methanol and potassium cyanide in the presence of crown ether.
  • Another method for cleaving the protected analog/peptide from the resin when the chloromethylated resin is employed is by ammonolysis or by treatment with hydrazine. If desired, the resulting C-terminal amide or hydrazide can be hydrolyzed to the free C-terminal carboxyl moiety, and the protecting groups can be removed conventionally.
  • the protecting group present on the N-terminal ⁇ -amino group may be removed preferentially either before or after the protected analog/peptide is cleaved from the support.
  • the analogs/peptides are substituted at their C-termini with cysteine.
  • a disulfide bond can be formed between the terminal cysteines, thereby crosslinking the analog/peptide chains.
  • disulfide bridges are conveniently formed by metal-catalyzed oxidation of the free cysteines or by nucleophilic substitution of a suitably modified cysteine residue. Selection of the crosslinking agent will depend upon the identities of the reactive side chains of the amino acids present in the analogs/peptides. For example, disulfide crosslinking would not be preferred if cysteine were present in the analog/peptide at additional sites other than the C-terminus. Also within the scope hereof are analogs/peptides crosslinked with methylene bridges.
  • Suitable crosslinking sites on the analogs/peptides aside from the N-terminal amino and C-terminal carboxyl groups, include epsilon amino groups found on lysine residues, as well as amino, imino, carboxyl, sulfhydryl and hydroxyl groups located on the side chains of internal residues of the analogs/peptides or residues introduced into flanking sequences.
  • Crosslinking through externally added crosslinking agents is suitably achieved, e.g., using any of a number of reagents familiar to those skilled in the art, for example, via carbodiimide treatment of the analog or peptide.
  • suitable multi-functional (ordinarily bifunctional) crosslinking agents are found in the literature.
  • Lys/Asp cyclization has been accomplished using Na-Boc-amino acids on solid-phase support with Fmoc/9-fluorenylmethyl (OFm) side-chain protection for Lys/Asp; the process is completed by piperidine treatment followed by cyclization.
  • OFm Fmoc/9-fluorenylmethyl
  • Disulfide crosslinked or cyclized analogs/peptides are generated by conventional methods.
  • the method of Pelton et al. J. Med. Chem., 29: 2370-2375 (1986) is suitable, except that a greater proportion of cyclo-oligomers are produced by conducting the reaction in more concentrated solutions than the dilute reaction mixture described by Pelton et al., for the production of cyclo-monomers.
  • the same chemistry is useful for synthesis of dimers or cyclo-oligomers or cyclo-monomers.
  • Also useful are thiomethylene bridges. Lebl and Hruby, Tetrahedron Letters, 25: 2067-2068 (1984). See also Cody et al., J. Med. Chem., 28: 583 (1985).
  • the desired cyclic or polymeric analogs/peptides are purified by gel filtration followed by reversed-phase high-pressure liquid chromatography or other conventional procedures.
  • the analogs/peptides are sterile filtered and formulated into conventional pharmacologically acceptable vehicles.
  • analogs/peptides may exist as diastereoisomers, enantiomers or mixtures thereof.
  • the syntheses described above may employ racemates, enantiomers or diastereomers as starting materials or intermediates. Diastereomeric products resulting from such syntheses may be separated by chromatographic or crystallization methods. Likewise, enantiomeric product mixtures may be separated using the same techniques or by other methods known in the art.
  • Each of the asymmetric carbon atoms when present, may be in one of two configurations R) or S) and both are within the scope of the present invention.
  • analogs and peptides of this invention may be contacted with the cartilage by any suitable technique, and may be combined, analog with analog, analog with peptide, or peptide with peptide.
  • the analog or peptide is administered to the mammal via, e.g., oral, parenteral (e.g., intramuscular, intraperitoneal, intravenous, intra-articular, or subcutaneous injection or infusion, or implant), nasal, pulmonary, vaginal, rectal, sublingual, or topical routes of administration, and can be formulated in dosage forms appropriate for each route of administration.
  • the specific route of administration will depend, e.g., on the medical history of the patient, including any perceived or anticipated side effects using the analog or peptide, the type of analog or peptide being administered, and the particular type of disorder to be corrected.
  • the administration is by continuous infusion (using, e.g., slow-release devices or minipumps such as osmotic pumps or skin patches), or by injection (using, e.g., intravenous, intra-articular or subcutaneous means).
  • the analog or peptide is administered locally, for example, directly to the joint where repair or prevention is needed.
  • the analog or peptide to be used in the therapy will be formulated and dosed in a fashion consistent with good medical practice, taking into account the clinical condition of the individual patient (especially the side effects of treatment with the analog or peptide), the type of disorder, the site of delivery, the method of administration, the scheduling of administration, and other factors known to practitioners.
  • the effective amounts of the analog or peptide for purposes herein are thus determined by such considerations and must be amounts that result in bioavailability of the drugs to the mammal and the desired effect.
  • a preferred administration is a chronic administration of about two times per day for 4-8 weeks to reproduce the effects of IGF-1.
  • chronic infusion may be employed using an infusion device for continuous subcutaneous (SC) or intra-articular infusions.
  • An intravenous bag solution may also be employed.
  • the key factor in selecting an appropriate dose for the disorder in question is the result obtained, as measured by criteria for measuring treatment of the cartilage disorder as are deemed appropriate by the medical practitioner.
  • the total pharmaceutically-effective amount of the analog or peptide administered parenterally per dose will be in a range that can be measured by a dose-response curve.
  • IGFs bound to IGFBPs or in the blood can be measured in body fluids of the mammal to be treated to determine the dosing.
  • the amount of analog or peptide to be employed can be calculated on a molar basis based on these serum levels of IGF-1 and IGF-2.
  • this method is carried out in vivo, i.e., after the fluid is extracted from a mammal and the IGF levels measured, the analog or peptide herein is administered to the mammal using single or multiple doses (that is, the contacting step is achieved by administration to a mammal). Then the IGF levels are re-measured from fluid extracted from the mammal.
  • Another method for determining dosing is to use antibodies to the analog or peptide or another detection method for the analog or peptide in the LIFA format. This would allow detection of endogenous or exogenous IGFs bound to IGFBP and the amount of analog or peptide bound to the IGFBP.
  • one method for detecting endogenous or exogenous IGF bound to an IGF binding protein or the amount of the analog or peptide herein or detecting the level of unbound IGF in a biological fluid. This method comprises:
  • (c) quantitatively analyzing the amount of the labeled means bound as a measure of the IGFBP in the biological fluid, and therefore as a measure of the amount of bound analog or peptide and IGF binding protein, bound IGF and IGF binding protein, or active IGF present in the fluid.
  • the amount of analog or peptide that may be employed can be estimated, i.e., from about 1 ⁇ g/kg/day to 10 mg/kg/day, preferably about 10 ⁇ g/kg/day to 1 mg/kg/day, more preferably about 10-200 ⁇ g/kg/day, might be used, based on kg of patient body weight, although, as noted above, this will be subject to a great deal of therapeutic discretion.
  • sustained-release compositions include semi-permeable polymer matrices in the form of shaped articles, e.g., films, or microcapsules.
  • Sustained-release matrices include polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman et al., Biopolymers, 22, 547-556 (1983), poly(2-hydroxyethyl methacrylate) (Langer et al., J. Biomed. Mater.
  • the liposomes are of the small (from or about 200 to 800 Angstroms) unilamellar type in which the lipid content is greater than about 30 mol. percent cholesterol, the selected proportion being adjusted for the most efficacious therapy.
  • the analog or peptide is formulated generally by mixing each at the desired degree of purity, in a unit dosage injectable form (solution, suspension, or emulsion), with a pharmaceutically, or parenterally, acceptable carrier, i.e., one that is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation.
  • a pharmaceutically, or parenterally, acceptable carrier i.e., one that is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation.
  • the formulation preferably does not include oxidizing agents and other peptides that are known to be deleterious to polypeptides.
  • the carrier suitably contains minor amounts of additives such as substances that enhance isotonicity and chemical stability.
  • additives such as substances that enhance isotonicity and chemical stability.
  • Such materials are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, succinate, acetic acid, and other organic acids or their salts; antioxidants such as ascorbic acid; low molecular weight (less than about ten residues) polypeptides, e.g., polyarginine or tripeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; glycine; amino acids such as glutamic acid, aspartic acid, histidine, or arginine; monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, mannose, trehalose, or dextrins; chelating agents such as EDTA; sugar alcohols such as
  • the analog or peptide typically formulated in such vehicles at a pH of from or about 4.5 to 8. It will be understood that use of certain of the foregoing excipients, carriers, or stabilizers will result in the formation of salts of the analog or peptide.
  • the final preparation may be a stable liquid or lyophilized solid.
  • the analog or peptide to be used for therapeutic administration must be sterile. Sterility is readily accomplished by filtration through sterile filtration membranes (e.g., 0.2 micron membranes). Therapeutic compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
  • a sterile access port for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
  • the analog or peptide ordinarily will be stored in unit or multi-dose containers, for example, sealed ampules or vials, as an aqueous solution or as a lyophilized formulation for reconstitution.
  • a lyophilized formulation 10-mL vials are filled with 5 mL of sterile-filtered 1% (w/v) aqueous solution of analog or peptide, and the resulting mixture is lyophilized.
  • the infusion solution is prepared by reconstituting the lyophilized analog or peptide using bacteriostatic Water-for-Injection.
  • Combination therapy with the analog or peptide herein and one or more other appropriate reagents that enhance the effect of the analog or peptide is also part of this invention.
  • these include antagonists to cytokines, NO, or IL-1ra, a cartilage catabolism antagonist, or a cartilage growth factor, such as wild-type IGF-1 and/or ALS if the active agent is an IGFBP-3 displacer peptide or an IGF-1 analog with a binding affinity preference for IGFBP-3 over IGFBP-1.
  • the IGFBP displacer peptide may be co-administered with an IGF-1 analog herein, preferably with the analog with a binding affinity preference for IGFBP-1 over IGFBP-3.
  • the invention herein also contemplates using gene therapy for treating a mammal, using nucleic acid encoding the analog or peptide.
  • gene therapy is used to increase (or overexpress) IGF levels in the mammal.
  • Nucleic acids that encode the analog or peptide can be used for this purpose. Once the amino acid sequence is known, one can generate several nucleic acid molecules using the degeneracy of the genetic code, and select which to use for gene therapy.
  • nucleic acid (optionally contained in a vector) into the patient's cells for purposes of gene therapy: in vivo and ex vivo.
  • in vivo delivery the nucleic acid is injected directly into the patient, usually at the site where the analog or peptide is required.
  • ex vivo treatment the patient's cells are removed, the nucleic acid is introduced into these isolated cells and the modified cells are administered to the patient either directly or, for example, encapsulated within porous membranes which are implanted into the patient. See, e.g., U.S. Pat. Nos. 4,892,538 and 5,283,187.
  • capsid proteins or fragments thereof tropic for a particular cell type
  • antibodies for proteins that undergo internalization in cycling and proteins that target intracellular localization and enhance intracellular half-life.
  • the technique of receptor-mediated endocytosis is described, for example, by Wu et al., J. Biol. Chem., 262: 4429-4432 (1987); and Wagner et al., Proc. Natl. Acad. Sci. USA, 87: 3410-3414 (1990).
  • Wu et al. J. Biol. Chem., 262: 4429-4432 (1987); and Wagner et al., Proc. Natl. Acad. Sci. USA, 87: 3410-3414 (1990).
  • the instruction on, or associated with, the container indicates that the composition is used for treating a cartilage disorder.
  • the instruction could indicate that the composition is effective for the treatment of osteoarthritis, rheumatoid arthritis, or any other degenerative cartilagenous disorder.
  • the article of manufacture may further comprise a second container comprising a pharmaceutically-acceptable buffer, such as phosphate-buffered saline, Ringer's solution, or dextrose solution.
  • the composition may contain any of the carriers, excipients, and/or stabilizers mentioned hereinabove. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.
  • the kit optionally includes a separate container, preferably a vial, for a co-agent to be administered along with the active agent, such as IGF-1.
  • Examples 1-4 below are taken from WO 98/45427 as describing IGFBP-3 displacer peptides defined herein. Based upon the results of in vitro and in vivo experiments using an IGFBP-3 displacer peptide with amino acid changes at residues 24 and 31 (Y24L,Y31A), also designated (Leu 24 ,Ala 31 ) hIGF-1 or IGF-M, disclosed in WO 98/45427, it is predicted that other peptides that inhibit the interaction of an IGF with an IGFBP, and bind poorly or not at all to the IGF-1 receptor, should increase active IGF levels in a subject being treated.
  • peptides that bind specifically and with measurable affinity to target molecules can be identified from an initial library of many binding and non-binding peptides through binding selections using bacteriophage coat-protein fusions (Smith, Science, 228: 1315 (1985); Scott and Smith, Science, 249: 386 (1990); Cwirla et al., Proc. Natl. Acad. Sci. USA, 8: 309 (1990); Devlin et al., Science, 249: 404 (1990); reviewed by Wells and Lowman, Curr. Opin. Struct. Biol., 2: 597 (1992); U.S. Pat. No. 5,223,409).
  • both proteins and peptides displayed on phage can be affinity-enhanced through iterative cycles of mutations, selection, and propagation.
  • Libraries of peptides differing in sequence at particular residue positions can be constructed using synthetic oligodeoxynucleotides. Peptides are displayed as fusion proteins with a phage coat protein (such as g3p or g8p) on bacteriophage particles, each of which contains a single-stranded DNA genome encoding the particular peptide variant. After cycles of affinity purification, using an immobilized target molecule, individual bacteriophage clones are isolated, and the amino acid sequence of their displayed peptides is deduced from their DNA sequences.
  • a phage coat protein such as g3p or g8p
  • Structural constraints or frameworks have previously been used for presentation of peptide libraries on phage and for subsequent, successive enhancement of binding affinities through mutation and selection. Such structured frameworks may favor stable binding conformations of peptide segments.
  • immunoglobulins provide a stable (and conserved) structural framework for presentation of a diversity of different peptide loops (CDR's, complementarity-determining regions) which can bind different antigens.
  • plasmid Used as a template for library constructions was a plasmid, pt4.g8 (complete DNA sequence shown in FIG. 1) expressing an antibody-recognizable (gD-tag) peptide fused to g8p of bacteriophage M13.
  • This plasmid contains single-stranded and double-stranded origins of DNA replication.
  • the phoA promoter and STII secretion-signal sequences are upstream of the gD peptide (underlined below), which is followed by a “linker” peptide (double underlined below), and then the g8p of bacteriophage M13:
  • N indicates a mixture of the nucleotides A, G, C, and T
  • S represents a mixture of the nucleotides G and C.
  • Unconstrained libraries i.e., having no fixed residues within the peptide have also yielded specific binding molecules (Scott and Smith, supra; Cwirla et al., supra; Devlin et al., supra; Kay et al., Gene, 128: 59 (1993)). Such libraries may yield structured peptides, nevertheless, since noncovalent interactions may still induce structure in the bound and/or unbound forms.
  • An unconstrained peptide library, of the form X 20 (SEQ ID NO:58), was constructed using oligonucleotide HL-302:
  • the number of transformants per library was approximately 1.8 ⁇ 10 8 for library HL-300, 7.9 ⁇ 10 8 for HL-301, 5.0 ⁇ 10 8 for HL-302, 5.3 ⁇ 10 8 for HL-303, 5.6 ⁇ 10 8 for HL-304, 5.0 ⁇ 10 8 for HL-305, 6.3 ⁇ 10 8 for HL-306, 4.5 ⁇ 10 8 for HL-307, 1.9 ⁇ 10 8 for HL-308, and 2.1 ⁇ 10 8 for HL-309.
  • IGFBP-3 and IGF-1 were biotinylated with a 1.5:1 molar ratio of a cleavable biotin reagent, EZ-LINKTM NHS-SS-Biotin (Pierce), to protein, using the manufacturer's instructions.
  • the blocking solution was then removed, and a solution of biotinylated target protein was added. After 1-2 h at room temperature, the target solution was removed, and the plates were washed ten times with PBS/TWEENTM surfactant (0.05% TWEEN-20TM in PBS buffer).
  • Phage from the libraries described above were pooled as follows: pool A consisted of HL-300 phage, pool B of HL-301 phage, pool C of HL-302 phage, and pool D of phage from the HL-303, HL-304, HL-305, HL-306, HL-307, HL-308, and HL-309 libraries. Phage were added in PBS/TWEENTM/albumin/biotin (PBS/TWEENTM buffer with 1 ⁇ M biotin, 5 g/l bovine serum albumin, or ovalbumin) to wells coated with each target, and with control wells that were coated with NEUTRAVIDINTM or with albumin, but not biotinylated target. The phage were allowed to bind 5-15 h at room temperature. The plates were then washed ten times with PBS/TWEENTM buffer.
  • PBS/TWEENTM buffer PBS/TWEENTM buffer with 1 ⁇ M biotin, 5 g/l
  • Phage remaining bound to the plates were eluted by incubating with 50 mM DTT for 1-2 h at room temperature.
  • the eluted phage were transfected into E. coli cells and allowed to grow overnight at 37° C. to amplify the phage.
  • the second and third cycles of binding selection were carried out as above, except that streptavidin (0.1 mg/ml) was included in the phage cocktails along with biotin. An aliquot was taken from each target-coated and control well incubated with each library, and serial dilutions of the diluted phage were performed to measure specific binding to target. The diluted phage were then transfected into E. coli cells and plated for colony counting.
  • the fourth round of binding selection was carried out on MAXISORPTM plates directly coated with 2 ⁇ g/ml of each target protein, or with albumin only.
  • the results of phage-binding selections in cycles 2-4 are shown in FIG. 2.
  • the same initial phage libraries (A, B, C, D) were also used for binding selections to directly-coated IGFBP-3.
  • MAXISORPTM 96-well plastic plates (Nunc) were coated with a solution of 2 ⁇ g/ml of IGFBP-3 in 50 mM sodium carbonate buffer, pH 9.6, overnight at 4° C. The target solution was then removed, and the plates were incubated with a blocking solution of 5 g/L of bovine serum albumin, for 1-2 h at room temperature. Phage were incubated with the plates as above, and non-binding phage washed away. The phage remaining bound were eluted by incubating with 20 mM HCl for 10 min at room temperature. Thereafter, the acid-eluted phage were neutralized with one-fifth volume of 1 M Tris-HCl, pH 8.0. Phage were transfected for colony counting as described above.
  • Peptide-phage clones were isolated by mixing phage pools with E. coli cells, and plating onto antibiotic-containing media. Colonies were isolated and grown with helper phage (as above) to obtain single-stranded DNA for sequencing. Peptide sequences selected for binding IGFBP-3 or IGF-1 were deduced from the DNA sequences of phagemid clones. A number of such clones are represented by the peptide sequences in Tables II and III, respectively.
  • Such peptide-phage clones could represent specific target-binding peptides which either do or do not block ligand (IGF-1 to IGFBP-3) binding, or any of a number of non-binding or background members of the selected pool. To distinguish among these possibilities, phage clones were tested for the ability to bind to IGFBP-3 in the presence and absence of IGF-1.
  • IGFBP-3 was coated directly onto MAXISORPTM plates as above. Phage from clonal cultures were mixed with IGF-1 (100 nM final concentration), and incubated with the immobilized IGFBP-3 for 1 hour at room temperature. The plates were then washed ten times, as above, and a solution of rabbit anti-phage antibody mixed with a goat-anti-rabbit conjugate of horseradish peroxidase was added. After an incubation of 1 hour at room temperature, the plates were developed with a chromogenic substrate, o-phenylenediamine (Sigma). The reaction was stopped with addition of 1 ⁇ 2 volume of 2.5 M H 2 SO 4 . Optical density at 490 nm was measured on a spectrophotometric plate reader.
  • FIG. 5 shows the results of a blocking assay of several phagemid clones derived from three rounds of DTT elution, followed by one round of HCl elution, as described above.
  • the phagemid clone was grown from a single colony overnight at 37° C. in a culture volume of 5 ml. The phage particles were precipitated and resuspended in 0.5 ml of PBS buffer. A 50-fold dilution of each phage solution was made into PBS/TWEENTM buffer, and the phage were incubated with or without 100 nM IGF-1 on an IGFBP-3-coated MAXISORPTM plate. As shown in FIG. 5, most clones were >40% inhibited for binding to IGFBP-3 at these phage concentrations, although clone 4D3.11 was only 5% inhibited under these conditions.
  • FIG. 6 shows the results of a blocking assay of several phagemid clones derived from three rounds of HCl elution, as described above.
  • the phagemid clone was grown from a single colony overnight at 37° C. in a culture volume of 5 ml.
  • the phage particles were prepared as described above.
  • most clones were >80% inhibited for binding to IGFBP-3 at these phage concentrations, although clones 23A3.3 and 23A3.5 were only about 20% inhibited under these conditions.
  • phage clones whose binding to an IGFBP-3 coated plate was inhibited only at low phage concentrations appear to yield higher-affinity peptides (see below) for IGFBP-3 than do those phage clones whose binding to an IGFBP-3 coated plate was inhibited both at high and at low phage concentrations (e.g., 4C3.2, 4D3.5, corresponding to peptides BP-23 and BP-24, respectively).
  • this type of phage-titration blocking assay may be generally useful as a means to predict the relative affinities and inhibitory potencies of peptides derived from phage displayed libraries.
  • peptide cDNAs from two round 4 g8 library pools, 4B and 4D were transferred to a g3 vector for monovalent phage display. Binding selections were carried out for three rounds, as described above, with acid elution of binding phage.
  • affinity improvements can be obtained by iteratively mutating, selecting, and propagating peptide-phage libraries, as described for hGH. See, e.g., U.S. Pat. No. 5,534,617.
  • Peptides were synthesized corresponding to a number of phage-derived sequences. In cases where two Cys residues were found in the peptide sequence, the disulfide (oxidized or “ox” suffix) monomeric form of the peptide was prepared and purified. In cases where four Cys residues were found, the ⁇ 1-4,2-3 ⁇ -disulfide form was prepared and purified.
  • IGF-1 was immobilized on a dextran chip for inhibition assays using a BIACORETM 2000 surface-plasmon-resonance device (BIAcore, Inc., Piscataway, N.J.) to measure free binding protein.
  • IGF-1 was biotinylated as described above, and injected over a chip to which streptavidin had been coupled (BIAcore, Inc.) to give 400 to 800 RU (response units) of immobilized IGF-1.
  • the IGF-1 showed no detectable dissociation over the time course of each experiment.
  • Serial dilutions of peptide were mixed with a constant concentration (40 nM) of IGFBP-3.
  • the results show a dose-response curve for each peptide's inhibition of IGFBP-3 binding to the chip.
  • the most effective inhibitors of IGFBP-3 binding tested were peptides BP3-01-ox (corresponding to phage clone 4D3.3), and a truncated form of this peptide, BP3-15 (see Table V).
  • a disulfide bond is formed between the two Cys residues of each 2-Cys containing peptide.
  • the two Cys* residues form a disulfide and the remaining two form a second disulfide.
  • peptides showed IC50's of 2 ⁇ M and 0.75 ⁇ M, respectively.
  • Other peptides such as BP3-4D3.11 (phage clone 4D3.11 from g8 display and 3Bi.1 from g3 display) showed inhibition with IC50's of ⁇ 10 ⁇ M.
  • FIG. 9 shows the inhibition of two IGFBP-3-selected peptides, BP3-01-ox and BP3-02-ox, for IGF-1 binding to an IGFBP-3 plate. In contrast, these peptides did not inhibit IGF-1 binding to an IGFBP-1 coated plate (FIG. 10).
  • BP3-01-ox has a lower affinity for the IGFBP than the other molecules tested in this assay, the fact that BP3-01-ox inhibits binding of IGFBP-3 to IGF-1 is, in itself, useful for various purposes, including for the LIFA and other assays noted above. Further, the KIRA assay only used IGF-1; it did not employ IGF-2, and BP3-01-ox was found to inhibit binding of IGFBP-3 to IGF-2 as noted in the competition assay described below.
  • IGF-2 was immobilized on a dextran chip for inhibition assays using a BIACORETM 2000 surface-plasmon-resonance device (BIAcore, Inc., Piscataway, N.J.) to measure free binding protein.
  • IGF-2 was biotinylated as described above, and injected over a chip to which streptavidin had been coupled (BIAcore, Inc.) to give approximately 1500 RU of immobilized IGF-2.
  • the IGF-2 showed no detectable dissociation over the time course of each experiment.
  • Serial dilutions of peptide were mixed with a constant concentration (20 nM) of IGFBP-3.
  • results show a dose-response curve for each peptide's inhibition of IGFBP-3 binding to IGF-2.
  • Peptides BP3-01-ox, BP3-14, BP3-15, and BP3-17 showed IC50's of 0.92 ⁇ M, 1.0 ⁇ M, 0.78 ⁇ M, and 5.1 ⁇ M, respectively.
  • these peptides inhibit the binding of IGFBP-3 both to IGF-1 and to IGF-2.
  • This Example tests an IGFBP-3-specific peptide, BP3-15, for its ability to block the binding of 125 I-IGF-1 in human serum.
  • Human serum was incubated with 125 I-IGF-1 ⁇ the peptide and the amount of tracer bound to IGFBPs via size-exclusion chromatography was measured.
  • Addition of the peptide resulted in an approximate 42% decrease in 125 I-IGF-1 associated with the 150-KD IGF/IGFBP-3/ALS complex and a 59% increase in the amount of free 125 I-IGF-1.
  • the peptide did not decrease 125 I-IGF-1 binding to the 44-KD IGFBPs (in fact, it slightly increased it), indicating that the peptide only competes with IGF-1 for binding to IGFBP-3.
  • WO 98/45427 published Oct. 15, 1998 discloses the preparation and characterization of the IGFBP-1 displacer peptide BP1-01 (CRAGPLQWLCEKYFG) (SEQ ID NO:26).
  • the kinetics of BP1-01 peptide variants were examined in a BIAcoreTM (BIAcore, Inc., Piscataway, N.J.) assay using IGFBP-1 covalently coupled via EDC/NHS (as described by the manufacturer) to a dextran chip.
  • Peptide BP1-01 displayed dissociation kinetics too rapid to measure.
  • BP1-02 the 19-mer variant (SEVGCRAGPLQWLCEKYFG) (SEQ ID NO:27) displayed measurable kinetics.
  • the association rate constant was 2.30 ⁇ 10 5 M ⁇ 1 sec ⁇ 1 and the dissociation rate constant was 5.03 ⁇ 10 ⁇ 2 sec ⁇ 1 .
  • the latter implies a half-life for peptide dissociation from IGFBP-1 of approximately 28 sec.
  • the association rate constant is moderately fast, consistent with the notion that the peptide may not undergo significant conformation change upon binding to IGFBP-1.
  • a second series of peptides made use of non-natural amino acids to probe whether other structural features such as an added methyl group at the alpha carbon, or an isomer (D-alanine) could affect peptide binding to IGFBP-1.
  • the potencies of these peptides were measured by biotinylated-IGFBP-1 ELISA assay, with the results shown in Table IX. These results confirm the importance of side chains L6, L9, W8, and Y13 in the binding of BP1-01 to IGFBP-1. Structural contributions are also suggested by the effects of substitutions at R2 and A3.
  • substitutions such as aib substitutions at G4, Q7, E11, K12, and F14, had little or no effect upon binding affinity.
  • Peptides including one or more of these substitutions may nevertheless by useful because non-natural amino acids often confer upon a peptide greater resistance to proteolysis (see Schumacher et al., Science, 271: 1854 (1996) and references therein). Such peptides may achieve a longer half-life in serum than those having only natural amino acids.
  • NNS codons were used to generate diverse peptide libraries as described above. Affinity selections were performed by solution binding of phage to biotinylated IGFBP-1 (prepared as described above) in solution to minimize avidity effects. A similar strategy was used for antibody-phage selections by Hawkins et al., J. Mol. Biol., 226: 889 (1992). For each round of selection, the target amount was reduced to select for enhanced affinity variants. Typically, 10 9 -10 10 purified phage were preblocked with MPBST (5% skim milk in PBS+0.05% TWEENTM 20) for 1 hr at room temperature and screened for binding to biotinylated target. Binding conditions are described below.
  • MPBST 5% skim milk in PBS+0.05% TWEENTM 20
  • Phage that bound to target were captured by incubating with streptavidin-magnetic beads (Promega Corp., Madison, Wis.) for 2-5 minutes at room temperature. After binding, the beads were washed with PBS-TWEENTM/MPBST ten times before eluting with 0.1 M HCl. The eluate was immediately neutralized with 1 ⁇ 3 volume of 1 M TRIS, pH 8.0. The eluted phage were propagated by infecting XL1 for the next selection cycle. Rounds 1, 2, 3 were carried out with 400 nM, 200 nM, and 20 nM target, respectively, with 1-h incubations. Round 4 was carried out with 4 nM target overnight. All binding reactions were performed at room temperature.
  • pH 0753 is a derivative of phGHam-g3 (Lowman et al., Biochemistry, 30: 10832-10838 (1991)) in which the additional XbaI site in the alkaline phosphatase promoter (PhoA) region has been deleted using the oligonucleotide 5′-AAA AGG GTA TGT AGA GGT TGA GGT-31 (SEQ ID NO:129).
  • the ligated vector pH 0753 containing the IGF-1 open reading frame was named pIGF-g3. It encodes for IGF-1 harboring the double mutation G1S-A70V fused to a fragment of the gene III protein (residues 249-406) from the E.
  • Immunosorbent plates (Nunc, MAXISORPTM, 96 wells) were coated with 100 ⁇ l/well of 1 ⁇ g/ml IGFBP-1 or IGFBP-3 in PBS buffer pH 7.2 at 4° C. overnight. The plates were then blocked with 0.5% TWEEN 20TM/pBS (also used as binding buffer) for 2 hours at room temperature (proteinaceous blocking agents like bovine serum albumin were avoided to prevent potential IGF or IGFBP contamination).
  • E. coli cells (XL1-Blue, Stratagene) freshly transformed with phagemid vector were grown overnight in 5 mL 2YT medium (Sambrook et al., supra) in the presence of M13-VCS helper phage (Stratagene).
  • Phage particles were harvested and resuspended in PBS buffer as described in Lowman, H. B., “Phage Display of Peptide Libraries on Protein Scaffolds,” in Cabilly, S. (ed.), Combinatorial Peptide Library Protocols (Humana Press Inc.: Totowa, N.J., 1998), pp. 249-264. Then phage concentrations were normalized to yield a maximal ELISA signal of 0.2-0.4 for each mutant (Lowman, in Cabilly, S. (ed.), supra).
  • Threefold serial dilutions of soluble competitor were prepared on non-absorbent microtiter plates (Nunc, F, 96 wells) with binding buffer (0.5% TWEEN 20TM/PBS) containing phage at the previously determined concentrations.
  • the dilution range of competitor protein extended over six orders of magnitude, starting at 5 ⁇ M for IGFBP-1 and 500 nM for IGFBP-3.
  • the plates containing immobilized target were washed with 0.05% TWEENTM/PBS buffer and subsequently incubated with 80 ⁇ l/well of the premixed phage-competitor solutions for 1 hour at room temperature.
  • Human IGFBP-1 was expressed in CHO cells and purified from the conditioned medium as described by Mortensen et al., Endocrinology, 138: 2073-2080 (1997). Recombinant human IGFBP-3 has also been cloned and expressed in mammalian cells (Wood et al., Mol. Endocrinology, 2: 1176-1185 (1988)). Purification from conditioned medium essentially followed the procedure described for IGFBP-1, with use of an IGF affinity column (Martin and Baxter, J. Biol. Chem., 261: 8754-8760 (1986)).
  • IGF-1 mutants were as described for the IGF-1 wild-type (Joly et al., Proc. Natl. Acad. Sci. USA, 95: 2773-2777 (1998)), but without transient overexpression of oxidoreductases.
  • the purification procedure was based on a previous protocol (Chang and Swartz, “Single-Step Solubilization and Folding of IGF-1 Aggregates from Escherichia coli ” In Cleland, J. L. (ed.), Protein Folding In Vivo and In Vitro (American Chemical Society, Washington, D.C., 1993), pp. 178-188), with minor adaptations.
  • Washed refractile bodies were resuspended at approximately 2 mg/ml in 50 mM CAPS (3-(cyclohexylamino)-1-propanesulfonic acid; Sigma) buffer pH 10.4 containing 2 M urea, 100 mM NaCl, 20% MeOH, and 2 mM DTT. This procedure combines solubilization of retractile bodies and subsequent oxidative refolding of IGF-1 mutants (Chang and Swartz, supra). After 3 hrs at room temperature the refolding solutions were filtered through microconcentrator membranes (Centricon, Amicon) with a molecular weight cut off of 50 kDa.
  • CAPS 3-(cyclohexylamino)-1-propanesulfonic acid
  • IGF-1 monomeric IGF-1
  • IGF-swap containing two non-native disulfides; Hober et al., Biochemistry, 31: 1749-1756 (1992); Miller et al., Biochemistry, 32: 5203-5213 (1993)
  • refolding solutions were acidified with 5% acetic acid and loaded on a DynamaxTM C18 semi-preparative HPLC column (Varian; 10.0 mm ID) at 4 ml/min.
  • the binding affinities of the IGF variants for IGFBP-1 and IGFBP-3 were determined using a BIACORETM-2000 real time kinetic interaction analysis system (Biacore, Inc., Piscataway, N.J.) to measure association (ka) and dissociation (kd) rates.
  • Carboxymethylated dextran biosensor chips (CM5, BIAcore Inc.) were activated with EDC (N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride) and NHS(N-hydroxysuccinimide) according to the supplier's instructions.
  • IGF mutants in 20 mM sodium acetate, pH 4.8 were injected onto the biosensor chip at a concentration of 50 ⁇ g/ml to yield approximately 450-600 RU's (resonance-responce units) of covalently-coupled protein. Unreacted groups were blocked with an injection of 1 M ethanolamine.
  • Kinetic measurements were carried out by injecting two-fold serial dilutions (starting at 1 ⁇ M) of either IGFBP-1 or IGFBP-3 in running buffer (PBS, 0.05% TWEEN 20TM, 0.1% ovalbumin, 0.1% sodium azide) at 25° C. using a flow rate of 20 ⁇ l/min.
  • E3A, G7A, L10A, F25A, and F49A showed a differential effect in binding IGFBP-1 versus IGFBP-3.
  • the relative IC 50 for IGFBP-1 differed by more than 4-fold from the one for IGFBP-3 (FIG. 14, Table XIII, relative specificity).
  • E3A and F49A showed the biggest relative specificity factors in this group. Alanine substitution of E3 had virtually no effect on IGFBP-3 affinity (1.4 fold), while binding to IGFBP-1 was weakened 34-fold. Even more dramatic, the affinity of F49A was reduced more than 100-fold for IGFBP-1 but only 3.6-fold for BP-3.
  • Residues G7, L10, and F25 appeared to be important for binding of both IGFBP's, although showing a more pronounced loss of affinity for IGFBP-1 than for IGFBP-3 when substituted by alanines. No significant specificity determinant for IGFBP-3 was identified, such as a mutant binding much tighter to IGFBP-1 than to IGFBP-3. However, mutations E9A, D12A, F23A, Y24A, T29A, S34A, and D45A had slightly larger (about 2-fold) effects on IGFBP-3 than on IGFBP-1 binding.
  • V11A, R36A, and P39A were tested because these variants had not been displayed correctly on phage, based upon the antibody recognition experiments (see above).
  • R36A and P39A showed wild-type kinetics for both binding proteins, whereas V11A showed a 5-fold reduction in affinity for both IGFBP-1 and IGFBP-3.
  • IGFBP-3 interaction was generally much less affected by the alanine substitutions than was the interaction with IGFBP-1, despite the fact that IGFBP-3 binds IGF-1 with approximately 10-fold higher affinity.
  • IGFBP-3 binds IGF-1 with approximately 10-fold higher affinity.
  • P63A no alanine mutant exhibited a >6-fold reduction in IGFBP-3 affinity (FIG. 14; Table XIII).
  • IGF-1 binds IGFBP-3 with 25-fold reduced affinity (Heding et al., supra). This naturally-occurring form of IGF-1 lacks the first three N-terminal residues and shows increased mitogenic potency, presumably due to its reduction in IGFBP-binding (Bagley et al., Biochem. J., 259: 665-671 (1989)). Since none of the first three amino acid side chains seem to contribute any energy to the binding of IGFBP-3 (Table I) but nevertheless des(1-3)-IGF-1 is compromised in IGFBP-3 binding, without being limited to any one theory, it is hypothesized that backbone interactions might be involved.
  • IGFBP-1 and IGFBP-3 binding epitopes on the surface of IGF-1 have been probed by alanine-scanning mutagenesis. Both binding epitopes are illustrated in FIG. 18. Individual IGF-1 side-chain interactions play a much more important role for binding to IGFBP-1 than to IGFBP-3.
  • Two major binding patches are found for IGFBP-1 (FIG. 18A). One is situated on the upper face of the N-terminal helix (composed of G7, L10, V11, L14, F25, 143, and V44) and one the lower face (composed of E3, T4, L5, F16, V17, and L54). These two binding patches are bridged by F49 and R50.
  • IGFBP-3 the binding epitope is more diffuse and has shifted to include G22, F23, and Y24 (FIG. 18B). Binding of IGFBP-3 is generally much less sensitive to alanine substitutions. In fact, the biggest reduction in affinity (apart from P63A, see below) is a 6-fold decrease seen for G7A. This result is interesting since IGFBP-3 binds with 10-fold higher affinity to IGF-1 than does IGFBP-1. Most probably, without limitation to any one theory, interactions originating from the IGF-1 main chain backbone are contributing to the binding of IGFBP-3. This hypothesis is further substantiated by the experiments with the Ala(1-3)-IGF mutant.
  • IGF-1 uses different binding modes to associate with IGFBP-1 and IGFBP-3: a few amino acid side-chain interactions are important for binding to IGFBP-1, while backbone interactions seem to play a major energetic role for binding to IGFBP-3.
  • the low protein abundance in monovalent phage display may disfavor aggregation and misfolding. Additionally, fusing IGF-1 to the truncated g3 phage protein might exert a stabilizing effect on the native structure of the peptide.
  • IGFBP-3 The levels of IGFBP-3 are positively regulated by IGF-1.
  • This class of binding proteins is generally less abundant than IGFBP-3, and its levels are negatively regulated by insulin (Bach and Rechler, supra; Clemmons, supra, 1997; Jones and Clemmons, supra).
  • IGFBP-specific variants of IGF-1 are obtained. Combination of several alanine mutations generates a variant that binds IGFBP-1 very weakly while retaining high-affinity binding of IGFBP-3.
  • the design of IGFBP-1 specific variants that no longer bind to IGFBP-3, can involve phage display of IGF-1 and the randomization of amino acids at specific positions (Cunningham et al., 1994, supra; Lowman and Wells, J. Mol. Biol., 243: 564-578 (1993)).
  • IGFBP-specific IGF-1 variants may be used diagnostically and therapeutically as described herein.
  • IGF-1 analogs are identified in which binding affinity to IGFBP-1, IGFBP-3, or both binding proteins, was reduced.
  • the total alanine-scanning mutagenesis of IGF-1 identified glutamic acid 3 (E3) and phenylalanine 49 (F49), as well as phenylalanine 16 (F16) and phenylalanine 25 (F25) to some degree, as specificity determinants for binding to IGFBP-1.
  • IGFBP-3 Further improved specificity for IGFBP-3 was likely to be attained by cumulative mutation of IGF-1, because the effects of point mutations are often additive with respect to their contribution to the free energy of binding (Wells, Biochemistry 29: 8509 (1990)). Therefore, a double mutant of IGF-1, E3A/F49A, was constructed by combining point mutations E3A and F49A in a single molecule. Although F16A showed a smaller IGFBP-specificity effect (Example 1 and Dubaquié and Lowman, supra), the double mutant F16A/F49A was also constructed.
  • Y31C containing a single putative unpaired cysteinyl thiol, to facilitate site-specific immobilization of IGF-1 for binding assays.
  • Y31C was chosen because it is outside the binding epitopes for IGFBP-1 and IGFBP-3 (Dubaquié and Lowman, supra). This immobilization technique ensures a uniform ligand population (Cunningham and Wells, J. Mol. Biol., 234: 554 (1993)) for binding by the injected analyte (i.e., IGF binding protein).
  • the advantage of this method over the previously-employed amine coupling is that the IGF-1 N-terminus is unblocked and free of any potential amine linkages to the chip matrix. This may be especially important for binding analysis of IGFBP-1, which is believed to interact with side chains of the IGF-1 N-terminus (Dubaquié and Lowman, supra).
  • Y31C displayed on phage showed wild-type-like affinities for both IGFBP-1 and IGFBP-3, supporting the notion that the region around residue 31 is important in receptor binding, but forms no contact with the binding proteins (Bayne et al., J. Biol. Chem., 264: 11004 (1988); Bayne et al., J. Biol. Chem., 265: 15648 (1989)).
  • KIRA Kinase Receptor Activation Assay
  • IGFBP-1 and IGFBP-3 binding affinities of these variants are set forth in Table XIV and in Dubaquie and Lowman, supra.
  • Table XVIII summarizes the relative affinities and specificities from BIACORETM measurements.
  • variant concentrations were roughly estimated at 13 nM (“high concentration) or 1.3 nM (“low concentration”), based on optical density measurements.
  • the signal obtained for each IGF variant was compared to that of a standard-dilution series of wild-type IGF-1, and reported in terms of an apparent IGF-1 concentration corresponding to the observed activity in the KIRA assay (FIGS. 20A and 20B). Although exact relative potencies were not measured, these results show that all tested mutants maintain the ability to activate the IGF type I receptor. TABLE XVIII Relative IGFBP-1 and IGFBP-3 Affinities of IGF-1 Variants.
  • IGFBP-1 IGFBP-3 Specificity IGF-1 K D (mutant)/ K D (mutant)/ Relative BP-1/ Variant K D (IGF-1)) K D (IGF-1)) Relative BP-3 G1S/A70V* 1.1 1.5 0.7 T4A* 6.9 2.1 3.3 V11A* 5.1 4.5 1.1 F16A* 25 6.9 3.6 F25A* 25 5.1 4.9 R36A* 1.1 0.8 1.4 P39A* 1.0 1.5 0.7 F49A* 70 4.2 16.7 F16A/F49A ND 65.6 ND
  • Table XVIII shows that, in addition to F49A, F16A and F25A are both substantially reduced in affinity for IGFBP-1, but less so for IGFBP-3. Both still retain biological activity based on KIRA assays (FIG. 20).
  • FIG. 22A shows a time course of the rate at which both molecules are cleared from the blood of the animals. As expected due to their decreased IGFBP affinities, both variants were cleared at a faster rate compared to wild-type human IGF-1. Interestingly, the double mutant (E3A/F49A) was cleared faster than the single mutant (F49A), correlating well with the respective affinities for the major binding protein in the serum, IGFBP-3 (Table XV).
  • FIG. 22B shows the tissue-to-blood ratio for the IGF variants in different organs.
  • mutants are expected to be efficacious in treating cartilage disorders, since the alanine-substituted mutants only weakly bind to IGFBP-1 and there is disregulation in IGFBP-3 present in arthritic disorders (Martel-Pelletier et al., supra). It would also be expected that BP3-01 and BP3-15 would also be efficacious for this purpose in view of their role in displacing IGFBP-3 (Lowman et al., supra, 1998; WO 98/45427).
  • Articular cartilage was aliquoted into MICRONICSTM tubes (approximately 55 mg per tube) and incubated for at least 24 hours in the above media. Control (media alone), wild-type IGF-1, E3A/F49A, or F49A was then added to each tube (to a final concentration of 40 or 400 ng/ml as indicated). The media was harvested and changed at various time points (0, 24, 48 and 72 hours).
  • the IGF-1 analogs significantly decreased nitric oxide release (FIG. 28). In addition, the IGF-1 analogs blocked induction of nitric oxide by IL-1 ⁇ (FIG. 29).
  • nitric oxide production correlates with a diseased state, and since nitric oxide appears to play a role in both the erosive and the inflammatory components of joint diseases, a protein or peptide that decreases nitric oxide production would likely be beneficial for the treatment of degenerative cartilagenous disorders.
  • in vivo animal models suggest that inhibition of nitric oxide production reduces progression of arthritis (Pelletier et al., Arthritis Rheum., 7: 1275-1286 (1998); van de Loo et al., Arthritis Rheum., 41: 634-646 (1998); Stivieroth and Frolich, Br. J. Rheumatol., 37: 246-257 (1998)).
  • nitric oxide has detrimental effects on chondrocytes as well as other cell types within the joint. Since inhibition of nitric oxide has been shown to inhibit progression of arthritis in animals, the effect of the IGF analogs on nitric oxide further suggests that the tested IGF analogs would be protective for joint tissues in vivo. Finally, these analogs or the IGFBP displacer peptides are expected to have anabolic effects on tissues, such as arthritic cartilage, which are otherwise IGF-1 resistant.
  • IGFBP-selective variants demonstrated a 700-fold and 80,000-fold apparent reduction in affinity for IGFBP-1, while preserving low nanomolar affinity for IGFBP-3, the major carrier of IGF-1 in plasma.
  • Both variants displayed wild-type-like potency in cellular receptor kinase assays, stimulated human cartilage matrix synthesis, and retained their ability to associate with ALS in complex with IGFBP-3.
  • the half-life of these variants is still determined by IGFBP-3, but their activity is no longer regulated by IGFBP-1.
  • pharmacokinetic parameters and tissue distribution of these two IGF-1 variants in rats differed from wild-type IGF-1 as a function of their IGFBP affinities.
  • the IGFBPs are generally thought to inhibit the biological activity of IGF-1 by sequestering the growth factor into high-affinity complexes and thereby preventing its receptor association (Jones and Clemmons, Endocr. Rev. 16: 3-34 (1995)).
  • the levels of IGFBP-3 (and IGFBP-4) were found to be increased in human inflammatory synovial fluid (Kanety et al., J. Rheumatol. 23: 815-818 (1996)). This change in IGFBP homeostasis is thought to contribute to the pathological condition by depriving cells from the IGF-1 survival signal.
  • IGF-1 molecules were generated with selectively reduced affinity for IGFBP-3, without altering activity on the IGF type I receptor.
  • IGFBP-3 is the major carrier of IGF-1 in serum, the half-life and biological distribution of such IGF-1 variants would presumably be drastically altered.
  • IGF-1 is an electrostatically polarized protein with a continuous negatively-charged patch at the N-terminus (including the B-region helix), while the C-region is mainly positively charged.
  • residue D12 was selected, since it does not contribute any binding energy for IGFBP-1 and seems to be part of the structural IGFBP-3 binding epitope (Dubaquié and Lowman, supra). It was reasoned that replacing residue 12 with a positive charge would disrupt the continuous negatively-charged patch that might possibly be involved in the IGFBP-3 interaction.
  • Matrix breakdown was determined by measuring the amount of proteoglycans in the media using the DMMB assay as set forth above.
  • Matrix (proteoglycan) synthesis was determined by measuring 35 S-sulfate uptake as set forth above.
  • F49A, E3A/F49A, F16A/F49A, D12K, D12R, or wild-type IGF-1 were tested for cartilage matrix synthesis in human tissue as described above.
  • Human articular cartilage from diseased joints was cultured in media alone or with F49A, E3A/F49A, F16A/F49A, D12K, D12R or wild-type IGF-1 (at 40 ng/ml) and matrix synthesis was determined by measuring 35 S-sulfate uptake as described above.
  • IGF-1 variants D12K and D12R are biologically active, as shown in the articular matrix synthesis stimulation experiments herein.
  • IGF-1 is a key regulator of matrix homeostasis in articular cartilage.
  • the metabolic imbalance in osteoarthritis that favors matrix breakdown over new matrix synthesis may be due, at least in part, to insensitivity of chondrocytes to IGF-1 stimulation. While the mechanism underlying this IGF-1 resistance is not known, without being limited to any one theory, it is believed that IGFBPs, which are elevated in many arthritic patients, play a role. In these patients, IGF-1 analogs that do not bind to, and are thus not inhibited by, IGFBPs would likely stimulate cartilage repair in tissue that is otherwise IGF-1 resistant.
  • Il-1 ⁇ has catabolic effects on cartilage, including the generation of synovial inflammation, up-regulation of matrix metalloproteinases, stimulation of matrix breakdown, and inhibition of proteoglycan and collagen synthesis. Furthermore, IL-1 protein is found in diseased, but not normal joints. Thus, the ability of the tested analogs to have positive effects on cartilage, as well as to counteract the deleterious effects of IL-1 ⁇ , strongly suggests that such molecules would have a protective effect on cartilage disorders, including damaged and/or diseased cartilage.
  • Human ALS was expressed in CHO cells (Leong et al., Mol. Endocrinol., 6: 870-876 (1992)). The secreted ALS was enriched on DEAE-Sepharose, followed by affinity purification on an IGF-1/IGFBP-3 column, as described in Baxter et al., J. Biol. Chem., 264: 11843-11848 (1989).
  • F49A and E3A/F49A Form Ternary Complexes with IGFBP-3 and ALS
  • the affinity of ALS for a crosslinked IGF-1/IGFBP-3 complex is on the order of 0.1 to 0.3 nM, depending on the carbohydrate content of ALS (Janosi et al., supra).
  • the dissociation rates of the wild-type IGF-1, F49A, and E3A/F49A remained constant between 4.0 ⁇ 10 ⁇ 4 s ⁇ 1 to 6.3 ⁇ 10 ⁇ 4 s ⁇ 1 (Table XIX).
  • the experiment confirms that both IGF-1 variants tested form ternary complexes with ALS.
  • IGFBP-3 in the blood seems to be saturated under normal conditions (Jones and Clemmons, Endocr. Rev., 16: 3-34 (1995)), it is believed, without limitation to any one theory, that the injected IGF-1 variants have to compete with endogenous IGF-1 for IGFBP-3 binding.
  • G4 was previously found to be substitutable by D-alanine. Because the conformational effects of D-alanine are different from those of L-alanine, L-alanine was substituted for G4 in peptide BP1-29. Inhibition assays showed a 50-fold loss in binding affinity with this substitution (Table XXI).
  • P5 was previously found to be highly conserved in phage-displayed peptide libraries; however, some substitutions were observed. For example, three different peptide-phage clones were found with arginine at this position. Therefore, the L-alanine substitution for proline was tested, as well as several alternative substitutions (BP1-30, BP1-31, BP1-34). The results (Table XXI) show that P5A, P5N, and P5R are well tolerated.
  • L6 and L9 were completely conserved in 40 of 40 sequenced clones and 61 of 61 sequenced clones, respectively, from two different IGFBP-1 selected peptide-phage libraries.
  • substitution of either of these residues with L-alanine or aib (alpha-aminoisobutyrate) side-chains resulted in a significant loss in IGFBP-1 binding affinity.
  • Two further substitutions were tested at each position: norleucine (Nle), an isomer of leucine, or arginine (the aliphatic portion of the side-chain of which might still be able to pack into the peptide structure).
  • the crosslinking chemistry involves replacement of the appropriate two residues with glutamic acid residues (the first and last Glu (E) residues shown in Table XXII), where the two Glu residues are joined by forming amides with 1,5-diaminopentane.
  • This cross-linking method has been described in WO 98/20036, supra.
  • Peptides were assayed in a BIAcoreTM assay as described in WO 98/45427, supra. These inhibition assays (FIG. 35) compared the relative potency of these peptides for blocking the interaction of IGFBP-1 with IGF-1. Adding the “i+7 helical lock” to a variant of BP1-01 reduced relative potency (Table XXII) by 6-fold (peptide (i+8)C) to 8-fold (peptide (i+7)D or (i+8)B) in the best locked-helix variants. These peptides demonstrate that a disulfide bond is not necessary to obtain structured, functional peptides of the BP1-01 family.
  • Peptide BP1-25 (Table XXV) was synthesized to test the additivity (Wells, Biochemistry, 29: 8509-8517 (1990)) for the N-terminal and C-terminal maximally-preferred substitutions. Compared with BP1-16 in inhibition assays, BP1-25 showed about a 20-fold affinity improvement. However, the affinity of BP1-25 was not significantly improved over BP1-21A. This affinity improvement was confirmed in other assays described below.
  • a monovalent-display peptide-phage library presenting BP1-21A as a fusion to g3p, was randomized (Lowman, Methods Mol. Biol., 87: 249-264 (1998)) at the N-terminal four residues.
  • Binding selection to IGFBP-1 was carried out by first allowing library phage to bind to solution biotinylated IGFBP-1, with an initial concentration of 50 nM, followed by 28 nM for the subsequent four rounds of selection.
  • Peptide-phage capable of binding IGFBP-1 were captured by incubating with streptavidin magnetic beads (Promega) for 10 minutes at room temperature.
  • the direct binding kinetics of IGFBP-1 peptides were measured by injecting a series of 2-fold diluted peptides in running buffer (0.05% TWEEN 20TM in PBS) over a carboxy-methyl (CM) biosensor chip coupled with about 590-1000 RU of IGFBP-1 at a flow rate of 50 ⁇ l/min on a BIAcore-2000TM or BIAcore-3000TM instrument.
  • CM carboxy-methyl
  • off-rate measurement was set for 30 minutes. This allowed for regeneration of IGFBP-1 on the chip by simple dissociation, rather than by addition of eluent.
  • a global fit of the sensorgram data was performed using a 1:1 Langmuir binding model. On-rates ranged from 4 ⁇ 10 5 to 1.9 ⁇ 10 6 M ⁇ 1 s ⁇ 1 .
  • the binding affinities, K D calculated as k off /k on are summarized in Table XXV.
  • Peptides BP1-20, BP1-21A, BP1-25, and BP1-40 were all found to have similar binding affinities (K D ) of about 20 nM to 40 nM.
  • N-terminal extensions to the BP1-01 peptide can improve binding affinity (as in BP1-02, BP1-20, BP1-21A, BP1-25, BP1-40, and other variants identified in Table XXIV). Some substitutions may alter expression levels in E. coli , since GQQS (SEQ ID NO:147) was clearly selected from phage-displayed peptide libraries. However, peptides having the sequences SEVG (SEQ ID NO:148), SEMV (SEQ ID NO:149), EARV (SEQ ID NO:150), or GQQS (SEQ ID NO:151) at their N-termini all had similar binding affinities. Therefore, the nature of added side-chains at the N-terminus appears to have little effect upon peptide binding affinity. This suggests that main-chain interaction of the peptide in this region may contribute to binding affinity for IGFBP-1.
  • a cell-based (KIRA) assay was previously described for measuring the amount of IGF-like activity displaced by peptides from mixtures of IGF-I and binding proteins (Lowman et al., supra, 1998; WO 98/45427, supra).
  • the KIRA assay was used to compare in vitro bioactivity of BP1-16, BP1-02, BP1-25, and BP1-40.
  • IGF-I and peptide were mixed and added to cells expressing IGF receptor for 30 min, then IGFBP-1 was added for an additional 1 h.
  • BP1-21A was designed for peptide biosynthesis in E. coli .
  • a DNA sequence encoding the peptide was fused by site-directed mutagenesis to the gene for a consensus domain of protein-A known as Z-domain (Nilsson et al., supra, 1987).
  • Z-domain a consensus domain of protein-A known as Z-domain
  • the fusion protein was enzymatically cleaved with trypsin to yield free peptide, which can be purified from the enzymatic reaction mix (see, e.g., Varadarajan et al., supra; Castellanos-Serra et al., supra; Nilsson et al., supra, 1996).
  • the fusion protein BP1-625-Z was produced from E. coli shake-flask cultures. Culture supernatants were sterile-filtered, then applied to an IgG-SepharoseTM column (Pharmacia). The bound fraction was eluted with 1M acetic acid, then lyophilized and resuspended in trypsin-digest buffer: 10 mM Tris (pH 8.0), 100 mM NaCl, 1 mM CaCl 2 . TPCK-treated trypsin (Sigma) was added at a weight/weight ratio of 1:100 to 1:200 (trypsin to substrate) and digestion was carried out at 25° C. for 1-2 hours.
  • peptide BP1-625 fraction was lyophilized and resuspended in 100 mM HEPES buffer, pH 7.2. Inhibition experiments were carried out in a BIAcoreTM assay as previously described, except that limiting amounts (9-10 nM IGFBP-1) were used to make the assay sensitive with respect to affinities in the 10 ⁇ 8 M range. These assays showed that the BP1-625 peptide blocked IGFBP-1 binding to immobilized IGF-1 and was similar in activity to BP1-25, having about 20-fold improved potency over BP1-01 (FIG. 38).
  • BP1-625 will block IGF-I binding to IGFBP-1 and produce IGF-like activity on cells, with similar potency to BP1-21A, BP1-25, or BP1-40. It would also be expected that a peptide, BP1-625T, comprising the sequence:
  • the BP1-625-Z fusion is useful for producing IGFBP-binding peptides from E. coli , and the Z part of the fusion can be advantageously attached to other peptides herein than just BP1-625.
  • IGFBPs The role of specific IGFBPs in IGF-1 activity was tested by treating articular cartilage explants from patients undergoing joint replacement with IGF-1 in the presence of peptides that inhibit IGF-1 binding to particular IGFBPs.
  • BP1-16 inhibits IGF-1 binding to IGFBP-1
  • BP3-15 inhibits IGF-1 binding to IGFBP-3
  • BP1-17 binds with much lower affinity to IGFBP-1.
  • buffer alone 100 mM HEPES
  • these three peptides, and especially BP3-15 and BP1-16, are expected to be useful therapeutics for the treatment of arthritis.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Veterinary Medicine (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Diabetes (AREA)
  • Immunology (AREA)
  • Epidemiology (AREA)
  • Toxicology (AREA)
  • Endocrinology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Physical Education & Sports Medicine (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Marine Sciences & Fisheries (AREA)
  • Biomedical Technology (AREA)
  • Orthopedic Medicine & Surgery (AREA)
  • Rheumatology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
US09/858,935 1997-04-04 2001-05-16 Method for treating cartilage disorders Abandoned US20030069177A1 (en)

Priority Applications (4)

Application Number Priority Date Filing Date Title
US09/858,935 US20030069177A1 (en) 2000-05-16 2001-05-16 Method for treating cartilage disorders
US10/271,869 US7423017B2 (en) 1997-04-04 2002-10-16 Method for treating cartilage disorders
US11/929,468 US7947650B2 (en) 1997-04-04 2007-10-30 Article of manufacture
US11/934,582 US8110548B2 (en) 1997-04-04 2007-11-02 Method for treating cartilage disorders

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US20449000P 2000-05-16 2000-05-16
US24898500P 2000-11-15 2000-11-15
US09/858,935 US20030069177A1 (en) 2000-05-16 2001-05-16 Method for treating cartilage disorders

Related Parent Applications (2)

Application Number Title Priority Date Filing Date
US09/337,227 Continuation-In-Part US6420518B1 (en) 1997-04-04 1999-06-22 Insulin-like growth factor agonist molecules
US47792300A Continuation-In-Part 1997-04-04 2000-01-05

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US10/271,869 Continuation US7423017B2 (en) 1997-04-04 2002-10-16 Method for treating cartilage disorders

Publications (1)

Publication Number Publication Date
US20030069177A1 true US20030069177A1 (en) 2003-04-10

Family

ID=26899527

Family Applications (4)

Application Number Title Priority Date Filing Date
US09/858,935 Abandoned US20030069177A1 (en) 1997-04-04 2001-05-16 Method for treating cartilage disorders
US10/271,869 Expired - Fee Related US7423017B2 (en) 1997-04-04 2002-10-16 Method for treating cartilage disorders
US11/929,468 Expired - Fee Related US7947650B2 (en) 1997-04-04 2007-10-30 Article of manufacture
US11/934,582 Expired - Fee Related US8110548B2 (en) 1997-04-04 2007-11-02 Method for treating cartilage disorders

Family Applications After (3)

Application Number Title Priority Date Filing Date
US10/271,869 Expired - Fee Related US7423017B2 (en) 1997-04-04 2002-10-16 Method for treating cartilage disorders
US11/929,468 Expired - Fee Related US7947650B2 (en) 1997-04-04 2007-10-30 Article of manufacture
US11/934,582 Expired - Fee Related US8110548B2 (en) 1997-04-04 2007-11-02 Method for treating cartilage disorders

Country Status (9)

Country Link
US (4) US20030069177A1 (fr)
EP (1) EP1282437B1 (fr)
AT (1) ATE389416T1 (fr)
AU (1) AU2001263215A1 (fr)
DE (1) DE60133271T2 (fr)
DK (1) DK1282437T3 (fr)
ES (1) ES2301547T3 (fr)
HK (1) HK1050321A1 (fr)
WO (1) WO2001087323A2 (fr)

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100137204A1 (en) * 2006-12-29 2010-06-03 Zheng Xin Dong Glp-1 pharmaceutical compositions
KR20110083648A (ko) * 2008-10-06 2011-07-20 가부시끼가이샤 쓰리디 매트릭스 조직 폐색제
US10245299B2 (en) 2014-03-10 2019-04-02 3-D Matrix, Ltd. Autoassembling peptides for the treatment of pulmonary bulla
US10369237B2 (en) 2014-03-10 2019-08-06 3-D Matrix, Ltd. Sterilization and filtration of peptide compositions
US10654893B2 (en) 2014-03-10 2020-05-19 3-D Matrix, Ltd. Self-assembling peptide compositions
US10793307B2 (en) 2012-07-06 2020-10-06 3-D Matrix, Ltd. Fill-finish process for peptide solutions
US10814038B2 (en) 2016-01-06 2020-10-27 3-D Matrix, Ltd. Combination compositions
US11324703B2 (en) 2017-12-15 2022-05-10 3-D Matrix, Ltd. Surfactant peptide nanostructures and uses thereof in drug delivery

Families Citing this family (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1099443A1 (fr) 1999-11-11 2001-05-16 Sulzer Orthopedics Ltd. Dispositif pour greffe/implant et sa méthode de production
US7387889B2 (en) * 2002-08-22 2008-06-17 Massachusetts Institute Of Technology Measurement of concentrations and binding energetics
US8173162B2 (en) * 2003-02-26 2012-05-08 Zimmer Orthobiologics, Inc. Preparation for repairing cartilage tissue, especially articular cartilage defects
EP1740190A1 (fr) * 2004-03-05 2007-01-10 Synthes GmbH Utilisation d'un melange pour produire un agent servant a traiter un cartilage defectueux ou degenere in vivo et pour produire un substitut de cartilage naturel in vitro
WO2005122723A2 (fr) * 2004-06-09 2005-12-29 Beth Israel Deaconess Medical Center Compositions et procedes d'amelioration de la reparation du cartilage articulaire
EP1764117A1 (fr) 2005-09-20 2007-03-21 Zimmer GmbH Implant pour la réparation de défauts cartilagineux et son procédé de préparation
AU2007212085B2 (en) 2006-02-07 2011-11-17 Spinalcyte, Llc Methods and compositions for repair of cartilage using an in vivo bioreactor
CL2007002502A1 (es) * 2006-08-31 2008-05-30 Hoffmann La Roche Variantes del factor de crecimiento similar a insulina-1 humano (igf-1) pegilados en lisina; metodo de produccion; proteina de fusion que la comprende; y su uso para tratar la enfermedad de alzheimer.
JP5739816B2 (ja) 2008-12-19 2015-06-24 バクスター・インターナショナル・インコーポレイテッドＢａｘｔｅｒ Ｉｎｔｅｒｎａｔｉｏｎａｌ Ｉｎｃｏｒｐ０Ｒａｔｅｄ Ｔｆｐｉインヒビターおよび使用法
WO2011115712A2 (fr) 2010-03-19 2011-09-22 Baxter International Inc Inhibiteurs de tfpi et procédés d'utilisation associés
US20140314726A1 (en) * 2011-11-09 2014-10-23 Spinalcyte, Llc Fibroblasts for treatment of degenerative disc disease
WO2013141965A1 (fr) 2012-03-21 2013-09-26 Baxter International Inc. Inhibiteurs de la voie du facteur tissulaire (tfpi) et procédés d'utilisation
US9637531B2 (en) * 2012-06-25 2017-05-02 The Brigham And Women's Hospital, Inc Selective cartilage therapy
ES2870558T3 (es) 2013-06-19 2021-10-27 Spinalcyte Llc Adipocitos para aplicaciones de condrocitos

Family Cites Families (87)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4411890A (en) * 1981-04-14 1983-10-25 Beckman Instruments, Inc. Synthetic peptides having pituitary growth hormone releasing activity
US3721252A (en) * 1971-04-01 1973-03-20 Catheter And Instr Corp Spring guide washer
US4444047A (en) * 1979-06-29 1984-04-24 Vdo Adolf Schindling A.G. Apparatus for determining the fuel consumption of injection internal combustion engines
EP0023469B1 (fr) 1979-07-16 1983-07-20 Ciba-Geigy Ag Post-traitement au moyen d'ammoniac liquide de matériaux cellulosiques fibreux apprêtes par un agent ignifugeant
US4511503A (en) * 1982-12-22 1985-04-16 Genentech, Inc. Purification and activity assurance of precipitated heterologous proteins
IL71991A (en) 1983-06-06 1994-05-30 Genentech Inc Preparation of human FGI and FGE in their processed form through recombinant AND tranology in prokaryotes
WO1985000831A1 (fr) 1983-08-10 1985-02-28 Amgen Expression microbienne d'un facteur de croissance semblable a l'insuline
JPH0720993B2 (ja) * 1985-08-22 1995-03-08 グロペップ プロプライエタリー リミテッド 生長因子
PH25772A (en) 1985-08-30 1991-10-18 Novo Industri As Insulin analogues, process for their preparation
SE8505922D0 (sv) 1985-12-13 1985-12-13 Kabigen Ab Construction of an igg binding protein to facilitate downstream processing using protein engineering
DE3881801T2 (de) 1987-04-06 1993-12-23 Celtrix Pharma Menschliche somatomedin-träger-protein-untereinheiten und verfahren zu ihrer herstellung.
ES2007309T3 (es) 1987-04-23 1994-11-01 Monsanto Co Secrecion del factor de crecimiento insulinoide 1 en e.coli.
SE8703625D0 (sv) * 1987-09-18 1987-09-18 Kabivitrum Ab New medical use
US4876242A (en) * 1987-09-21 1989-10-24 Merck & Co., Inc. Human insulin-like growth factor analoges with reduced binding to serum carrier proteins and their production in yeast
US5470828A (en) * 1987-12-24 1995-11-28 Gropep Pty. Ltd. Peptide analogs of insulin-like growth factor II
DE3852636T2 (de) 1987-12-24 1995-05-04 Gropep Pty Ltd Peptid-analoge vom insulinähnlichen wachstums-faktor 1 (igf-1) oder faktor 2 (igf-2).
DE68905203T2 (de) * 1988-02-05 1993-07-22 Ciba Geigy Ag Verwendung von igf i zur herstellung eines praeparates fuer die behandlung von nierenkrankheiten.
US4988675A (en) * 1988-02-05 1991-01-29 Ciba-Geigy Corporation Method for preventing secondary effects
DK131988A (da) 1988-03-11 1989-09-12 Erasmus University Igf-bindingsprotein, dna-struktur, der koder for igf-bindingsproteinet og vektor indeholdende denne dna-struktur
US5258287A (en) 1988-03-22 1993-11-02 Genentech, Inc. DNA encoding and methods of production of insulin-like growth factor binding protein BP53
JPH03504597A (ja) 1988-04-12 1991-10-09 シナージェン，インコーポレーテッド インスリン様成長因子活性の強化および阻害方法
US5223409A (en) * 1988-09-02 1993-06-29 Protein Engineering Corp. Directed evolution of novel binding proteins
US5750373A (en) * 1990-12-03 1998-05-12 Genentech, Inc. Enrichment method for variant proteins having altered binding properties, M13 phagemids, and growth hormone variants
US5534617A (en) * 1988-10-28 1996-07-09 Genentech, Inc. Human growth hormone variants having greater affinity for human growth hormone receptor at site 1
US6780613B1 (en) * 1988-10-28 2004-08-24 Genentech, Inc. Growth hormone variants
GB8826451D0 (en) 1988-11-11 1988-12-14 Sandoz Ltd Improvements in/relating to organic compounds
IL92816A0 (en) 1988-12-22 1990-09-17 Biogrowth Inc Recombinant dna molecules,hosts,processes and human somatomedin carrier protein-like polypeptides
CA2007886A1 (fr) 1989-01-17 1990-07-17 Marvin L. Bayne Analogues de facteur de croissance humain ressemblant a l'insuline avec liaison reduite aux proteines de liaison 28 k igf et leur production dans des levures
US5514646A (en) * 1989-02-09 1996-05-07 Chance; Ronald E. Insulin analogs modified at position 29 of the B chain
US5093317A (en) * 1989-06-05 1992-03-03 Cephalon, Inc. Treating disorders by application of insulin-like growth factor
US5652214A (en) * 1989-06-05 1997-07-29 Cephalon, Inc. Treating disorders by application of insulin-like growth factors and analogs
GB8920381D0 (en) 1989-09-08 1989-10-25 Greater Glasgow Health Board Treatment of insulin-resistant diabetes
SE500903C2 (sv) 1989-12-20 1994-09-26 Atlas Copco Constr & Mining Bergborrningsrigg
ZA9010332B (en) 1989-12-22 1991-08-28 Ciba Geigy Method of reducing or preventing adverse effect of steroid therapy and compositions therefor
NZ236618A (en) 1990-01-03 1997-06-24 Ciba Geigy Ag Treating and preventing osteoporosis using insulin-like growth factor i (igf i) in conjunction with a bone antiresorptive active compound
US5126324A (en) * 1990-06-07 1992-06-30 Genentech, Inc. Method of enhancing growth in patients using combination therapy
US5374620A (en) * 1990-06-07 1994-12-20 Genentech, Inc. Growth-promoting composition and its use
US5364839A (en) 1990-06-18 1994-11-15 Genetics Institute, Inc. Osteoinductive pharmaceutical formulations
US5210017A (en) * 1990-11-19 1993-05-11 Genentech, Inc. Ligand-mediated immunofunctional hormone binding protein assay method
US5593844A (en) 1990-11-19 1997-01-14 Genentech, Inc. Ligand-mediated immunofunctional hormone binding protein assay method
SE9100099D0 (sv) 1991-01-11 1991-01-11 Kabi Pharmacia Ab Use of growth factor
US5206023A (en) 1991-01-31 1993-04-27 Robert F. Shaw Method and compositions for the treatment and repair of defects or lesions in cartilage
US5187151A (en) * 1991-02-12 1993-02-16 Genentech, Inc. Use of binding protein with igf-i as an anabolic growth promoting agent
CA2105300C (fr) 1991-03-01 2008-12-23 Robert C. Ladner Procede pour le developpement de mini-proteines fixatrices
US5206235A (en) * 1991-03-20 1993-04-27 Merck & Co., Inc. Benzo-fused lactams that promote the release of growth hormone
US5202119A (en) * 1991-06-28 1993-04-13 Genentech, Inc. Method of stimulating immune response
DE69218948T2 (de) * 1991-08-01 1997-07-31 Auckland Uniservices Ltd IGF-I zur Verbesserung der neuronale Lage
US6310040B1 (en) 1991-11-08 2001-10-30 Cephalon, Inc. Treating retinal neuronal disorders by the application of insulin-like growth factors and analogs
TW267102B (fr) 1992-03-13 1996-01-01 Ciba Geigy
SE9201573D0 (sv) 1992-05-19 1992-05-19 Kabi Pharmacia Ab Use of igf-1
ES2140463T3 (es) * 1992-06-12 2000-03-01 Cephalon Inc Prevencion y tratamiento de la neuropatia periferica.
GB9217696D0 (en) 1992-08-20 1992-09-30 Agricultural & Food Res Use of specific binding molecules
US5273961A (en) * 1992-09-22 1993-12-28 Genentech, Inc. Method of prophylaxis of acute renal failure
US5342763A (en) * 1992-11-23 1994-08-30 Genentech, Inc. Method for producing polypeptide via bacterial fermentation
WO1994016722A1 (fr) 1993-01-25 1994-08-04 The Beth Israel Hospital Association Procede de modification, de diagnostic et de depistage de caracteristiques de barriere cellulaire sensibles a l'igf-i
EP0686040A1 (fr) 1993-01-29 1995-12-13 Amgen Boulder Inc. Composition de cicatrisation de plaies
US5478852C1 (en) 1993-09-15 2001-03-13 Sankyo Co Use of thiazolidinedione derivatives and related antihyperglycemic agents in the treatment of impaired glucose tolerance in order to prevent or delay the onset of noninsulin-dependent diabetes mellitus
ATE202785T1 (de) 1993-12-23 2001-07-15 Novo Nordisk As Verbindungen mit wachstumshormon-freisetzenden eigenschaften
IL112112A (en) 1993-12-23 2000-06-29 Novo Nordisk As Peptides with growth hormone releasing properties and pharmaceutical compositions containing them
US5597700A (en) * 1994-04-28 1997-01-28 California Research, Llc Method for detecting free insulin-like growth-factor-binding protein 1 and a test device for detecting the ruptures of fetal membranes using the above method
US5444047A (en) 1994-06-16 1995-08-22 Dipasquale; Gene Treatment of arthritic and post-surgical orthopedic conditions with Insulin-like Growth Factor-I
SE9402331D0 (sv) * 1994-07-01 1994-07-01 Pharmacia Ab New use
SE9402370D0 (sv) 1994-07-04 1994-07-04 Pharmacia Ab Use of IGF-I
US5712249A (en) * 1994-09-08 1998-01-27 Ciba-Geigy Corporation Use of insulin-like growth factors I and II for inhibition of inflammatory response
US5798337A (en) 1994-11-16 1998-08-25 Genentech, Inc. Low molecular weight peptidomimetic growth hormone secretagogues
US5596844A (en) * 1995-02-03 1997-01-28 Kalinowski; Juan R. Foldable portable building
SE9501472D0 (sv) 1995-04-21 1995-04-21 Pharmacia Ab Truncated IGF-I
US5622932A (en) 1995-05-05 1997-04-22 Eli Lilly And Company IGF-1 superagonists
US5565428A (en) * 1995-05-22 1996-10-15 Genentech, Inc. Method of administration of IGF-I
US5741776A (en) * 1995-05-22 1998-04-21 Genentech, Inc. Method of administration of IGF-I
PT828758E (pt) 1995-05-26 2002-02-28 Theratechnologies Inc Analogos quimericos de corpo gordo-pro-grf com potencia biologica aumentada
ZA964814B (en) 1995-06-07 1998-02-09 Glaxo Group Ltd Peptides and compounds that bind to a receptor.
AU6046696A (en) 1995-06-07 1996-12-30 Glaxo Group Limited Peptides and compounds that bind to a receptor
US5958872A (en) 1996-04-01 1999-09-28 Apoptosis Technology, Inc. Active survival domains of IGF-IR and methods of use
AU2676297A (en) 1996-04-17 1997-11-07 Neurocrine Biosciences, Inc. Ligand inhibitors of insulin-like growth factor binding proteins and methods of use therefor
AR048563A1 (es) * 1996-09-16 2006-05-10 Univ Dalhousie Metodos para el tratamiento de la enfermedad renal poliquistica en un mamifero, la insuficiencia renal, el desarrollo glomerular y de los rinones y proteger de la toxicidad por hormonas esteroides y uso de la igf- i para la fabricacion de una composicion farmaceutica.
EP0938497B1 (fr) 1996-11-06 2007-02-28 Genentech, Inc. Peptide a structure helicoidale contrainte et leurs procedes de fabrication
US6428781B1 (en) 1996-12-27 2002-08-06 Daiichi Pharmaceutical Co., Ltd. Composition of an endogenous insulin-like growth factor-II derivative
US6121416A (en) * 1997-04-04 2000-09-19 Genentech, Inc. Insulin-like growth factor agonist molecules
ZA984697B (en) 1997-06-13 1999-12-01 Lilly Co Eli Stable insulin formulations.
DE19757250A1 (de) 1997-12-22 1999-07-01 Forssmann Wolf Georg Prof Dr Insulin-like growth factor binding protein und seine Verwendung
JP2002510646A (ja) * 1998-04-03 2002-04-09 カイロン コーポレイション 関節軟骨障害を処置するためのｉｇｆｉの使用
ATE303159T1 (de) 1998-10-02 2005-09-15 Celtrix Pharma Null igf zur krebsbehandlung
WO2000023469A2 (fr) 1998-10-16 2000-04-27 Musc Foundation For Research Development Fragments du facteur de croissance proche de l'insuline et de la proteine de fixation du facteur de croissance proche de l'insuline, et utilisations de ces fragments
DK1141015T3 (da) * 1999-01-06 2010-01-25 Genentech Inc Insulin-lignende vækstfaktor (IGF) I-mutantvarianter
WO2000069901A2 (fr) 1999-05-19 2000-11-23 Xencor, Inc. Nouvelles proteines a action insulinoide utilisees dans le traitement du diabete
WO2001072771A2 (fr) 2000-03-29 2001-10-04 Dgi Biotechnologies, L.L.C. Agonistes et antagonistes du recepteur de l'insuline et d'igf-1

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100137204A1 (en) * 2006-12-29 2010-06-03 Zheng Xin Dong Glp-1 pharmaceutical compositions
US10576123B2 (en) * 2008-10-06 2020-03-03 3-D Matrix, Ltd. Tissue occluding agent comprising an IEIKIEIKIEIKI peptide
KR101670099B1 (ko) 2008-10-06 2016-10-27 가부시끼가이샤 쓰리디 매트릭스 조직 폐색제
US11801281B2 (en) 2008-10-06 2023-10-31 3-D Matrix, Ltd. Tissue occluding agent comprising an ieikieikieiki peptide
KR20110083648A (ko) * 2008-10-06 2011-07-20 가부시끼가이샤 쓰리디 매트릭스 조직 폐색제
US10596225B2 (en) 2008-10-06 2020-03-24 3-D Matrix, Ltd. Tissue occluding agent comprising an IEIKIEIKIEIKI peptide
US20110201541A1 (en) * 2008-10-06 2011-08-18 3-D Matrix, Ltd. Tissue occluding agent
US10793307B2 (en) 2012-07-06 2020-10-06 3-D Matrix, Ltd. Fill-finish process for peptide solutions
US12006085B2 (en) 2012-07-06 2024-06-11 3-D Matrix, Ltd. Fill-finish process for peptide solutions
US10654893B2 (en) 2014-03-10 2020-05-19 3-D Matrix, Ltd. Self-assembling peptide compositions
US11090398B2 (en) 2014-03-10 2021-08-17 3-D Matrix, Ltd. Sterilization and filtration of peptide compositions
US10369237B2 (en) 2014-03-10 2019-08-06 3-D Matrix, Ltd. Sterilization and filtration of peptide compositions
US10245299B2 (en) 2014-03-10 2019-04-02 3-D Matrix, Ltd. Autoassembling peptides for the treatment of pulmonary bulla
US10814038B2 (en) 2016-01-06 2020-10-27 3-D Matrix, Ltd. Combination compositions
US11324703B2 (en) 2017-12-15 2022-05-10 3-D Matrix, Ltd. Surfactant peptide nanostructures and uses thereof in drug delivery

Also Published As

Publication number Publication date
US20030211992A1 (en) 2003-11-13
HK1050321A1 (en) 2003-06-20
AU2001263215A1 (en) 2001-11-26
DE60133271D1 (de) 2008-04-30
US20090011988A1 (en) 2009-01-08
EP1282437A2 (fr) 2003-02-12
ES2301547T3 (es) 2008-07-01
ATE389416T1 (de) 2008-04-15
WO2001087323A2 (fr) 2001-11-22
DK1282437T3 (da) 2008-06-30
US8110548B2 (en) 2012-02-07
EP1282437B1 (fr) 2008-03-19
WO2001087323A3 (fr) 2002-04-11
US7423017B2 (en) 2008-09-09
US20100130411A1 (en) 2010-05-27
DE60133271T2 (de) 2009-04-23
US7947650B2 (en) 2011-05-24

Similar Documents

Publication Publication Date Title
US7947650B2 (en) Article of manufacture
US6949349B1 (en) Insulin-like growth factor agonist molecules
EP1141014B1 (fr) Variant mutant du facteur de croissance insulinomimetique (igf) de type i
US6506874B1 (en) IGF-I variants
AU2003236454B2 (en) Insulin-like growth factor (IGF) I mutant variants
CA2702760C (fr) Variants mutants du facteur de croissance i semblable a l'insuline (igf)
EP1506972A1 (fr) Variants mutants du facteur de croissance insulinomimétique (IGF) de type I

Legal Events

Date Code Title Description
AS Assignment

Owner name: GENETECH, INC., CALIFORNIA

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:DUBAQUIE, YVES;FILVAROFF, ELLEN;LOWMAN, HENRY B.;REEL/FRAME:011829/0217;SIGNING DATES FROM 20010628 TO 20010713

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION