EP1220909A2 - Compositions et methodes therapeutiques utilisant des proteines morphogeniques, des hormones et des recepteurs d'hormones - Google Patents

Compositions et methodes therapeutiques utilisant des proteines morphogeniques, des hormones et des recepteurs d'hormones

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Publication number
EP1220909A2
EP1220909A2 EP00965478A EP00965478A EP1220909A2 EP 1220909 A2 EP1220909 A2 EP 1220909A2 EP 00965478 A EP00965478 A EP 00965478A EP 00965478 A EP00965478 A EP 00965478A EP 1220909 A2 EP1220909 A2 EP 1220909A2
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European Patent Office
Prior art keywords
bmp
xaa
hormone
res
protein
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German (de)
English (en)
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John C. Lee
Lee-Chuan C. Yeh
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Stryker Corp
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Stryker Corp
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/204—IL-6
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/32—Bones; Osteocytes; Osteoblasts; Tendons; Tenocytes; Teeth; Odontoblasts; Cartilage; Chondrocytes; Synovial membrane
    • 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
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/1793—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • 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
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1875—Bone morphogenic factor; Osteogenins; Osteogenic factor; Bone-inducing factor
    • 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
    • A61K38/39—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological 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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system

Definitions

  • TGF- ⁇ Transforming Growth Factor-Beta
  • This superfamily includes osteogenic proteins ("OPs”) and bone morphogenic proteins ("BMPs").
  • OPs and BMPs share a highly conserved, bioactive cysteine-rich domain near their C-termini and have a propensity to form homo- and hetero-dimers.
  • Many morphogenic proteins belonging to the BMP family have been described. Some were isolated using purification techniques on the basis of osteogenic activity. Others were identified and cloned by virtue of DNA sequence homologies within conserved regions that are common to the BMP family.
  • BMPs are referred to as consecutively numbered BMPs whether or not they have demonstrable osteogenic activity. While several of the earliest members of the BMP family were identified by virtue of their ability to induce new cartilage and bone, a number of other BMPs have different or additional tissue-inductive capabilities. For example, BMP- 12 and BMP- 13 (identified by DNA sequence homology) reportedly induce tendon/ligament-like tissue formation in vivo (WO 95/16035). Several BMPs, including some of those originally isolated on the basis of their osteogenic activity, can induce neuron proliferation and promote axon regeneration (WO 95/05846; Liem et al., Cell, 82, pp. 969-79 (1995)). Thus, it appears that BMPs may have a variety of potential tissue-inductive capabilities whose final expression depends on a complex set of developmental and environmental cues.
  • Implantable osteogenic devices comprising mammalian osteogenic protein for promoting bone healing and regeneration have been described (see, e.g., Oppermann et al., U. S. Patent No. 5,354,557).
  • Some osteogenic devices contain porous, biocompatible matrices that allow the diffusion of osteogenic proteins into the implantation site as well as the influx and efflux of progenitor cells
  • Osteogenic protein- coated prosthetic devices that enhance the bond strength between the prosthesis and existing bone have also been described (Rueger et al , U S Patent No 5,344,654)
  • this invention features a method for improving the tissue inductive capability of a morphogenic protein at a target locus in a mammal
  • the morphogenic protein and a first effective amount of a hormone and a second effective amount of a soluble receptor of the hormone are administered to the target locus, wherein the morphogenic protein is capable of inducing tissue formation when accessible to a progenitor cell in the mammal, and the hormone and the receptor in combination enhance that capability
  • the morphogenic protein, hormone and hormone receptor can be administered simultaneously to the target locus Alternatively, the three components are administered separately, in any order for instance, the morphogenic protein can be administered first, and then the hormone and hormone receptor are administered together, or the morphogenic protein and the hormone are administered together first, and then the hormone receptor is administered
  • the morphogenic protein is administered via a nucleic acid (e g , a plasmid, a viral vector, or naked DNA) that comprises a sequence encoding the morphogenic protein and is capable of expressing
  • the morphogenic protein may comprise a pair of subunits disulfide-bonded to produce a dimeric species, wherein at least one of the subunits comprises a polypeptide belonging to the BMP protein family
  • the morphogenic protein may comprise an amino acid sequence sufficiently duplicative of the amino acid sequence of a reference BMP such as BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7 (OP-1), BMP-8, BMP-9, BMP-10.
  • the morphogenic protein is a homo- or heterodimer comprising a BMP-2 or BMP-7 (OP-1) subunit
  • the morphogenic protein is capable of inducing tissue formation For instance, it may be capable of inducing the progenitor cell to form tissue tendon/ gament- hke or neural-like tissue, or it may be an osteogenic protein that is capable of inducing the progenitor cell to form endochondral or intramembranous bone, or cartilage
  • the method of this invention thus can be used to induce tissue regeneration or repair in a variety of tissue defects such as bone, cartilage, soft tissue and neural tissue defects
  • Hormones useful in this invention include but are not limited to cytokines (e g , interleukins 1 through 18), growth factors (e g , fibroblast growth factor, vascular endothehal growth factor, platelet-derived growth factor, TGF- ⁇ , or prostaglandin) or morphogenic proteins
  • the invention also features pharmaceutical compositions and kits comprising a hormone and a soluble receptor thereof for improving the tissue inductive activity of a morphogenic protein
  • This invention also provides implantable morphogenic devices for inducing tissue formation in allogeneic and xenogeneic implants Such devices comprise a morphogenic protein, a hormone and a soluble receptor thereof disposed within a carrier
  • Methods for inducing local tissue formation from a progenitor cell in a mammal using those compositions and devices are also provided
  • a method for accelerating allograft repair in a mammal using those morphogenic devices is provided
  • This invention also provides a prosthetic device comprising a prosthesis coated with a morphogenic protein, a hormone and a soluble receptor thereof, and a method for promoting m vivo integration of an implantable prosthetic device to enhance the bond strength between the prosthesis and the existing target tissue at the joining site
  • Methods for treating tissue degenerative conditions in a mammal using the pharmaceutical compositions are also provided Unless otherwise
  • Fig. 1 is a bar graph showing that a combination of interleukin 6 (“IL-6”) and soluble IL-6 receptor (“sIL-6R”) significantly increases the ability of OP-1 to induce alkaline phosphatase (“AP”) activity in fetal rat calvaria (“FRC”) cells "OP” stands for “OP-1", “IL-6R “ refers to “sIL-6R " The parenthesized numbers indicate the protein concentrations (ng/ml) used in the assay, in the case of IL-6/sIL-6R combinations, the two numbers separated by a backslash in the parenthesis indicate the protein concentrations of IL-6 and sIL-6R, respectively
  • Fig 2 is a photograph showing results of a mineralized bone nodule formation assay using OP- 1 and IL-6 Dark spots inside the wells represent mineralized bone nodules
  • Fig 3 is a photograph showing results of a mineralized bone nodule formation assay using OP-1, IL-6 and sIL-6R Dark spots inside the wells represent mineralized bone nodules
  • Fig 4 is a bar graph showing the mRNA levels of BMPR-IA, BMPR-IB, ActR-I, and BMPR-II in various test groups "sR" stands for sIL-6R Values in the graph represent the means ⁇ SE of twelve Northern blots with RNA isolated from two different FRC cell preparations
  • Fig 5 is a bar graph showing that the AP activity in FRC cells transfected with the OP-1 -encoding pW24 plasmid is enhanced by exogenous sIL-6R alone or a combination of IL-6 and sIL-6R ("IL-6/R")
  • IL-6/R stands for sIL-6R Values in the graph represent the mean ⁇ SE of five independent determinations with three different FRC cell preparations and two different DNA preparations
  • IL-6/R (X/Y) refers to X ng/ml IL-6 and Y ng/ml sIL-6R
  • biocompatible refers to a material that does not elicit detrimental effects associated with the body's various protective systems, such as cell and humoral- associated immune responses, e g , inflammatory responses and foreign body fibrotic responses This term also implies that no specific undesirable effects, cytotoxic or systemic, are caused by the material when it is implanted into the patient
  • BMP refers to a protein belonging to the BMP family of the TGF- ⁇ superfamily of proteins defined on the basis of DNA and amino acid sequence homology
  • a protein belongs to the BMP family when it has at least 50% (e g , at least 70% or even 85%) amino acid sequence homology with a known BMP family member within the conserved C-terminal cysteine- ⁇ ch domain that characterizes the BMP family Members of the BMP family may have less than 50% DNA or ammo acid sequence homology overall
  • morphogenic protein refers to a protein having morphogenic activity
  • this protein is capable of inducing progenitor cells to proliferate and/or to initiate differentiation pathways that lead to the formation of cartilage, bone, tendon, ligament, neural or other types of tissue, depending on local environmental cues
  • morphogenic proteins useful in this invention may behave differently in different surroundings
  • a morphogenic protein of this invention may comprise at least one polypeptide belonging to the BMP family
  • osteoogenic protein refers to a morphogenic protein that is capable of inducing a progenitor cell to form cartilage and/or bone
  • the bone may be intramembranous bone or endochondral bone
  • Most osteogenic proteins are members of the BMP family and are thus also BMPs However, the converse may not be true
  • a BMP identified by sequence homology must have demonstrable osteogenic or chondrogenic activity in a functional bioassay to be an osteogenic protein
  • morphogenic activity refers to the ability of an agent to stimulate a target cell to undergo one or more cell divisions (proliferation) that may optionally lead to cell differentiation
  • target cells are referred to gene ⁇ cally herein as progenitor cells
  • Cell proliferation is typically characterized by changes in cell cycle regulation and may be detected by a number of means which include measuring DNA synthetic or cellular growth rates
  • Early stages of cell differentiation are typically characterized by changes in gene expression patterns relative to those of the progenitor cell, such changes may be indicative of a commitment towards a particular cell fate or cell type
  • Later stages of cell differentiation may be characterized by changes m gene expression patterns, cell physiology and morphology Any reproducible change in gene expression, cell physiology or morphology may be used to assess the initiation and extent of cell differentiation induced by a morphogenic protein
  • hormone/receptor pair refers to a combination of a hormone and a soluble receptor thereof
  • the hormone e g , a cytokine, a growth factor, or a morphogenic protein
  • a soluble receptor of a hormone is a compound that binds specifically to the hormone, and can, for example, be a polypeptide containing only the hormone-binding domain (e g , an extracellular domain) of a native cellular receptor of the hormone, an antibody specific for the hormone, or a chemical compound that specifically interacts with the hormone
  • a soluble receptor can also be a compound (e g , a protein) containing a domain that specifically binds to the hormone and another domain that specifically binds to the native cellular receptor of the hormone such that the
  • the morphogenic proteins of this invention are capable of stimulating a progenitor cell to undergo cell division and/or differentiation They may belong to the TGF- ⁇ protein superfamily, and include, but are not limited to, OP-1, OP-2, OP-3, BMP-2, BMP-3, BMP-3b, BMP-4, BMP-5, BMP-6, BMP-9, BMP-10, BMP-1 1, BMP-12, BMP-13, BMP-14, BMP-15, GDF-1, GDF-2, GDF-3, GDF-5, GDF-6, GDF-7, GDF-8, GDF-9, GDF-10, GDF-11, GDF-12, DPP, Vg-1, Vgr-1, 60 A protein, NODAL, UNIVIN, SCREW, ADMP, NEURAL, and TGF- ⁇
  • One of the preferred morphogenic proteins is OP-1 Nucleotide and amino acid sequences for hOP-1 are provided in SEQ ID NOs 1 and 2, respectively For ease of description, hOP-1 is recited as
  • useful morphogenic proteins also include polypeptides having at least 50% (e g , at least 70% or even 85%) sequence homology with a known morphogenic protein, particularly with a known BMP within the conserved C-terminal cysteine- ⁇ ch domain that characterizes the BMP protein family
  • morphogenic proteins include biologically active variants of any known morphogenic protein, including variants containing conservative ammo acid changes
  • useful morphogenic proteins include those containing sequences that share at least 70%) ammo acid sequence homology with the C-terminal seven-cysteine domain of hOP-1, which domain corresponds to the C- terminal 102-106 amino acid residues of SEQ ID NO 2
  • the C-terminal 102 ammo acid residues corresponds to residues 330-431 of SEQ ID NO 2
  • the morphogenic protein consists of a pair of subunits disulfide-bonded to produce a dimer, wherein at least one of the subunits comprises a recombinant polypeptid
  • amino acid sequence homology is understood to include both amino acid sequence identity and similarity Homologous sequences share identical and/or similar amino acid residues, where similar residues are conservative substitutions for, or "allowed point mutations" of, corresponding amino acid residues in an aligned reference sequence
  • a candidate polypeptide sequence that shares 70% ammo acid homology with a reference sequence is one in which any 70% of the aligned residues are either identical to, or are conservative substitutions of, the corresponding residues in a reference sequence
  • Certain particularly preferred morphogenic polypeptides share at least 60%) (e g , at least 65%) amino acid sequence identity with the C-termmal seven-cysteine domain of human OP-1
  • conservative substitutions are residues that are physically or functionally similar to the corresponding reference residues That is, a conservative substitution and its reference residue have similar size, shape, electric charge, chemical properties including the ability to form covalent or hydrogen bonds, or the like
  • Preferred conservative substitutions are those fulfilling the criteria defined for an accepted point mutation in Dayhoff et al , Atlas of Protein Sequence and Structure 5 345-352 (1978 & Supp )
  • Examples of conservative substitutions are substitutions within the following groups (a) valine, glycine, (b) glycine, alanine, (c) valine, isoleucine, leucine, (d) aspartic acid, glutamic acid, (e) asparagine, glutamine, (f) se ⁇ ne, threomne, (g) lysine, argirune, methionme, and (h) phenylalamne, tyro sine
  • the term "conservative variant” or “conservative variation” also includes
  • Amino acid sequence homology can be determined by methods well known in the art For instance, to determine the percent homology of a candidate ammo acid sequence to the sequence of the seven-cysteme domain, the two sequences are first aligned The alignment can be made with, e g , the dynamic programming algorithm described in Needleman et al , J Mol Biol 48 443 (1970), and the Align Program, a commercial software package produced by DNAstar, Inc The teachings b ⁇ both sources are incorporated by reference herein An initial alignment can be refined by comparison to a multi-sequence alignment of a family of related proteins Once the alignment is made and refined, a percent homology score is calculated The aligned amino acid residues of the two sequences are compared sequentially for their similarity to each other Similarity factors include similar size, shape and electrical charge One particularly preferred method of determining amino acid similarities is the PAM250 matrix described in Dayhoff et al , supra A similarity score is first calculated as the sum of the aligned pairwise amino acid similarity scores Insert
  • Morphogenic proteins useful herein include any known naturally occurring native proteins, including allelic, phylogenetic counterparts and other variants thereof These variants include forms having varying glycosylation patterns, varying N-termini, and active truncated or mutated forms of a native protein
  • Useful morphogenic proteins also include those that are biosynthetically produced (e g , "mutems or "mutant proteins") and those that are new, morphogenically active members of the general morphogenic family of proteins
  • Particularly useful sequences include those comprising the C-terminal 96 to 102 amino acid residues of DPP (from Drosoph ⁇ a), Vg-1 ( ⁇ r m Xenopus), Vgr-1 (from mouse), the OP1 and OP2 proteins (U S Patent No 5,011,691), as well as the proteins referred to as BMP-2, BMP-3, BMP-4 (WO 88/00205, U S Patent No 5,013,649 and WO 91/18098), BMP-5
  • Generic Sequence 7 (SEQ ID NO 4) and Generic Sequence 8 (SEQ ID NO 5), disclosed below, accommodate the homologies shared among preferred protein family members identified to date, including OP-1, OP-2, OP-3, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, 60A, DPP, Vg-1, Vgr-1, and GDF-1
  • the amino acid sequences for these proteins are described herein and/or in the art
  • the generic sequences include the identical amino acid residues shared by these sequences in the C-terminal six- or seven-cysteine skeletal domains (represented by Generic Sequences 7 and 8, respectively), as well as alternative residues for the variable positions within the sequences
  • the generic sequences provide an appropriate cysteine skeleton where inter- or intra-molecular disulfide bonds can form Those sequences contain certain specified amino acids that may influence the tertiary structure of the folded proteins
  • the generic sequences allow for an additional cysteine at position 36 (Generic Sequ
  • Generic Sequences 9 (SEQ ID NO 6) and 10 (SEQ ID NO 7) are composite amino acid sequences of the following proteins human OP-1 ("hOP-1"), hOP-2, hOP-3, hBMP-2, hBMP-3, hBMP-4, hBMP-5, hBMP-6, hBMP-9, hBMPIO, hBMP-11, Drosophila 60A, Xenopus Vg- 1 , sea urchin UNIVIN, hCDMP- 1 (mouse GDF-5 or
  • Generic Sequence 9 accommodates the C-terminal six-cysteine skeleton and, like Generic Sequence 8, Generic Sequence 10 accommodates the C-terminal seven-cysteine skeleton GENERIC SEQUENCE 9
  • useful proteins include active proteins comprising dimers having the generic amino acid sequence "OPX" (SEQ ID NO 3), which defines the seven-cysteine skeleton and accommodates the homologies between several identified variants of OP-1 and OP-2
  • OPX generic amino acid sequence
  • Each Xaa in OPX is independently selected from the residues occurring at the corresponding position in the C-terminal sequence of mouse or human OP-1 or OP-2
  • the morphogenic proteins comprise species of the generic amino acid sequence
  • each of the amino acids arranged vertically at each position in the sequence may be used alternatively in various combinations (SEQ ID NO 10)
  • SEQ ID NO 10 amino acids arranged vertically at each position in the sequence may be used alternatively in various combinations
  • useful morphogenic proteins comprise an amino acid sequence encoded by a nucleic acid that hybridizes, under low, medium or high stringency hybridization conditions, to DNA or RNA encoding reference morphogenic protein coding sequences
  • Exemplary reference sequences include the C-terminal sequences defining the conserved seven-cysteine domains of OP-1, OP-2, BMP-2, BMP-4, BMP-5, BMP-6, 60A, GDF-3, GDF-5, GDF-6, GDF-7, and the like
  • High stringent hybridization conditions are herein defined as hybridization in 40% formamide, 5X SSPE, 5X Denhardt's Solution, and 0.1% SDS at 37°C overnight, and washing in 0 IX SSPE, 0 1% SDS at 50°C Standard stringency conditions are well characterized in commercially available, standard molecular cloning texts See, for example, Molecular Cloning, A Laboratory Manual , 2nd Ed , ed by Sambrook et al.
  • Suitable in vitro, ex vivo and in vivo bioassays known in the art, including those described herein, may be used to ascertain whether a new BMP-related gene product has a morphogenic activity
  • Expression and localization studies defining where and when the gene is expressed may also be used to identify potential morphogenic activities
  • Nucleic acid and protein localization procedures are well known to those of skill in the art (see, e g , Ausubel et al , eds Current Protocols in Molecular Cloning, Greene Publishing and Wiley Interscience, New York, 1989)
  • Many of the identified BMPs are osteogenic and can induce bone and cartilage formation when implanted into mammals
  • Some BMPs identified based on sequence homology to known osteogenic proteins possess other morphogenic activities and a combination of a hormone and a soluble receptor thereof may be used to enhance those activities
  • BMP- 12 and BMP- 13 reportedly induce ectopic formation of tendon/ligament-like tissue when implanted into mammals
  • BMPs which are known to be osteogenic can also induce neuronal cell differentiation Embryonic mouse cells treated with BMP-2 or OP- 1 differentiate into astrocyte-like (glial) cells, and peripheral nerve regeneration using BMP-2 has been reported (Wang et al , WO 95/05846)
  • BMP-4, BMP-5 and OP-1 are expressed in epidermal ectoderm flanking the neural plate
  • Ectopic recombinant BMP-4 and OP-1 proteins can induce neural plate cells to initiate dorsal neural cell fate differentiation (Liem et al , Cell, 82, pp 969-79 (1995))
  • OP-1 and other BMPs can induce neural crest cell differentiation
  • OP-1 and these BMPs can induce many or all dorsal neural cell types, including roof plate cells, neural crest cells, and commissural neurons, depending on localized positional cues
  • osteogenic proteins originally derived from bone matrix are involved in neural development suggests that these and other members of the BMP family have additional tissue inductive properties that are not yet disclosed
  • hormone/receptor combinations set forth in this invention can be used to enhance new or known tissue inductive properties of various known morphogenic proteins
  • the invention described herein will be useful for stimulating tissue inductive activities of new morphogenic proteins as they are identified in the future Production of Morphogenic Proteins
  • the morphogenic proteins of this invention can be derived from a variety of sources For instance, they may be isolated from natural sources, recombinantly produced, or chemically synthesized
  • the morphogenic proteins of the invention can be purified from tissue sources, e g , mammalian tissue sources, using well known techniques See, e g ,
  • the morphogenic protein is produced by expressing an appropriate recombinant DNA molecule in a host cell
  • the DNA and amino acid sequences of many BMPs and OPs have been reported, and methods for their recombinant production are published and otherwise known to those of skill in the art
  • the homologous sequences may be cloned and sequenced using standard recombinant DNA techniques With the DNA sequence available, a DNA fragment encoding the morphogenic protein may be inserted into an expression vector selected to work in conjunction with a desired host expression system The DNA fragment is cloned into the vector such that its transcription is controlled by a heterologous promoter in the vector, preferably a promoter which may be optionally regulated
  • Useful host cells include but are not limited to bacteria such as E. coli, yeasts such as Saccharomyces and Picia, insects cells and other primary, transformed or immortalized eukaryotic cultured cells
  • Preferred eukaryotic host cells include CHO, COS and BSC cells (see below)
  • An appropriate vector is selected according to the host system selected
  • Useful vectors include but are not limited to plasmids, cosmids, bacte ⁇ ophage, insect and animal viral vectors, including those derived from retroviruses and other single and double- stranded DNA viruses
  • the morphogenic protein may be derived from a recombinant DNA molecule expressed in a prokaryotic host Using recombinant DNA techniques, various fusion genes have been constructed to induce recombinant expression of naturally sourced osteogenic sequences inE coli (see, e g ,
  • the morphogenic protein is expressed using a mammalian host-vector system (e g , transgemc production or tissue culture production)
  • a mammalian host-vector system e g , transgemc production or tissue culture production
  • a morphogenic protein so expressed may resemble more closely the naturally occurring protein While the glycosylation pattern of the recombinant protein may sometimes differ from that of the natural protein, such differences are often not essential for biological activity of the recombinant protein.
  • Techniques for transfection, expression and purification of recombinant proteins are well known in the art See, e g , Ausubel et al , supra, and Bendig, Genetic Engineering, 7, pp 91-127 (1988)
  • Mammalian DNA vectors should include appropriate sequences to promote expression of the gene of interest Such sequences include transcription initiation, termination and enhancer sequences, efficient RNA processing signals such as splicing and polyadenylation signals, mRNA-stabilizing sequences, translation-enhancing sequences (e g , Kozak consensus sequence), protein-stabilizing sequences, and when desired, sequences that enhance protein secretion
  • Useful promoters include, but are not limited to, the SV40 early and late promoters, the adenovirus major late promoter, the mouse metallothionein-I (“mMT”) promoter, the Rous sarcoma virus (“RS V”) long terminal repeat (“LTR”), the mouse mammary tumor virus (“MMTV”) LTR, and the human cytomegalovirus ("CMV”) major intermediate-early promoter
  • mMT mouse metallothionein-I
  • RS V Rous sarcoma virus
  • LTR Rous sarcoma virus
  • MMTV mouse mammary tumor virus
  • CMV human cytomegalovirus
  • Preferred DNA vectors also include a marker gene (e g , neomycin or
  • DHFR dihydrofolate reductase
  • MTX cytotoxic drug methotrexate
  • MTX cytotoxic drug methotrexate
  • Gene amplification can be further enhanced by modifying marker gene expression regulatory sequences (e g , enhancer, promoter, and transcription or translation initiation sequences) to reduce the levels of marker protein produced Lowering the level of DHFR transcription increases the DHFR gene copy number (and the physically- associated gene) to enable the transfected cell to adapt to growth in even low levels of methotrexate (e g , 0 1 ⁇ M MTX) Preferred expression vectors such as pH754 and pH752 (Oppermann et al , U S Patent No.
  • marker gene expression regulatory sequences e g , enhancer, promoter, and transcription or translation initiation sequences
  • Another gene amplification scheme relies on the temperature sensitivity (ts) of BSC40-tsA58 cells transfected with an SV40 vector Temperature reduction to 33°C stabilizes the temperature-sensitive SV40 T antigen, which leads to the excision and amplification of the integrated transfected vector DNA, thereby amplifying the physically- associated gene of interest
  • COS Monkey kidney cells
  • COS cells expressing the gene of interest can be established by transfectmg the cells with, e g , an SV40 vector carrying the gene Stably transfected cell lines, on the other hand, can be used for long term production of morphogenic proteins
  • both CHO cells and BSC40-tsA58 cells can be used as host cells
  • Recombinant OP-1 has been expressed in three different cell expression systems COS cells for rapidly screening the functionality of the various expression constructs, CHO cells for the establishment of stable cell lines, and BSC40-tsA58 cells as an alternative means of producing recombinant OP-1 protein
  • BMP-2, BMP-4, BMP-6 and BMP-7 (OP-1) - originally isolated from bone - are bioactive as either homodimers or heterodimers
  • OPs and BMPs are bioactive as either homodimers or heterodimers
  • the ability of OPs and BMPs to form heterodimers may confer additional or altered morphogenic activities on morphogenic proteins
  • Heterodimers may exhibit qualitatively or quantitatively different binding affinities than homodimers for OP and BMP receptors
  • Altered binding affinities may in turn result in differential activation of receptors that mediate different signalling pathways, ultimately leading to different biological activities
  • Altered binding affinities can also be manifested in a tissue or cell type-specific manner, thereby inducing only particular progenitor cell types to undergo proliferation and/or differentiation
  • the dime ⁇ c proteins can be isolated from the culture media and/or refolded and dime ⁇ zed in vitro to form biologically active compositions
  • Heterodimers can be formed in vitro by combining separate, distinct polypeptide chains
  • heterodimers can be formed in a single cell by co-expressing nucleic acids encoding separate, distinct polypeptide chains See, e g , WO 93/09229 and U S Patent No 5,411,941, for exemplary protocols for heterodimer protein production C.
  • the morphogenic protein of the invention can also be produced in vivo in a patient
  • an expression vector comprising a promoter operatively linked to a coding sequence of the morphogenic protein may be introduced into progenitor cells in the patient
  • a nucleic acid construct according to this invention is derived from a non- rephcating linear or circular DNA or RNA vector, or from an autonomously replicating plasmid or viral vector Alternatively, the construct is integrated into the host genome Any vector that can transfect or transduce the desired progenitor cell may be used
  • Preferred vectors are viral vectors, including those derived from replication-defective retroviruses (see, e g , WO89/07136, Rosenberg et al , N Eng J Mecl 323(9) 570-578 (1990)), adenovirus (see, e g , Morsey et al , J Cell Biochem , Supp 17E (1993)), adeno- associated virus (Kotin et al , Proc Natl Acad Sci USA 87 221 1-2215 (1990)), replication-defective herpes simplex viruses (HSV, Lu et al , Abstract, page 66, Abstracts of the Meeting on Gene Therapy
  • expression control sequences are operably linked to the nucleic acid sequence encoding the morphogenic protein useful in this invention
  • expression control sequences may include a promoter, an enhancer, such as one derived from an immunoglobulin gene, SV40, cytomegalovirus, etc , and a polyadenylation sequence
  • a nucleic acid construct of this invention may also contain an internal ⁇ bosome entry site ("IRES"), and an intron that may be desirably located between the promoter/enhancer sequence and the morphogenic protein-coding sequence Selection of these and other common vector elements are conventional See, e.g , Sambrook et al, supra, Ausubel et al , Current Protocols in Molecular Biology, John Wiley & Sons, New York, (1989), and references cited therein
  • promoters for this purpose include, without limitation, the retroviral Rous sarcoma virus (RSV
  • nucleic acid constructs of this invention may be formulated as a pharmaceutical composition for use in any form of transient and/or stable gene transfer in vivo and in vitro
  • the composition comprises at least the nucleic acid construct and a pharmaceutically acceptable carrier such as saline
  • a pharmaceutically acceptable carrier such as saline
  • Other aqueous and non-aqueous sterile suspensions known to be pharmaceutically acceptable carriers and well known to those of skill in the art may be employed also
  • the construct may be used for in vivo and ex vivo gene therapy, in vitro protein production and diagnostic assays
  • the nucleic acid construct can be introduced into target cells as naked DNA, or by, e g , liposome fusion (see, e.g , Nabel et al , Science 249.1285-8 (1990), Ledley, J Pediatrics 1 10 1-8 and 167-74 (1987), Nicolau et al , Proc Natl Acad Sci USA 80 1068-72 (1983)), erythrocyte ghosts, or microsphere methods (microparticles, see. e g , United States patent 4,789,734, United States patent 4,925,673, United States patent 3,625,214, Grego ⁇ adis, Drug Carriers in Biology and Medicine, pp 287-341, Academic Press, 1979)
  • the nucleic acid construct is viral-based, it can also be packaged as a vi ⁇ on which then is used to transduce a cell (e g , an autologous T cell isolated from a patient) in viti o
  • a cell e g , an autologous T cell isolated from a patient
  • the recombinant virus may be administered to a patient directly, e g , locally at the tissue defect site, or intravenously, lntrape ⁇ toneally, intranasally, intramuscularly, subcutaneously, and/or intradermally, as determined by one skilled in the gene therapy art
  • a slow-release device such as an implantable pump, may be used to facilitate delivery of the recombinant virus to a cell
  • the specific cells to be infected may be targeted by controlling the method of delivery
  • the treatments of the invention may be repeated as needed, as determined by one skilled in the art
  • an effective human dosage of a BMP-codmg virus is generally in the range of from about 0 5 ml to 50 ml of saline solution containing the virus at concentrations of about 1 x 10 7 , 1 x 10 8 , 1 x 10 9 , 1 x 10 10 , 1 x 10 u , 1 x 10 12 , 1 x 10' ⁇ 1 x 10 14 , 1 x 10 15 , or 1 x 10 16 viral particles per dose administered
  • the dosage will be adjusted to balance the corrective benefits against any adverse side effects
  • the levels of expression of BMP may be monitored to determine the type and frequency of dosage administration
  • a morphogenic protein may be prepared synthetically Morphogenic proteins prepared synthetically may be native, or may be non-native proteins, I e , those not otherwise found in nature
  • Non-native morphogenic proteins can be made by mutating native morphogenic proteins Methods for making mutations that favor refolding and/or assembling subunits into forms that exhibit greater morphogenic activity have been described See, e g , U S Patent No 5,399,677 Non-native morphogenic proteins can also be synthesized using a series of consensus sequences (U S Patent No 5,324,819) These consensus sequences were designed based on partial amino acid sequence data obtained from native osteogenic products and on their homologies with other proteins reportedly having a presumed or demonstrated developmental function Several biosynthetic consensus sequences (called consensus osteogenic proteins or "COPs”) have been expressed as fusion proteins in prokaryotes Purified fusion proteins may be cleaved, refolded, combined with a hormone and a soluble receptor thereof, implanted in an established animal model and examined for their bone- and/or cartilage-inducing activity Certain preferred synthetic osteogenic proteins comprise one or both of two synthetic amino acid sequences designated COP5 (
  • the morphogenic protein is a synthetic osteogenic protein comprising a partial or complete sequence of a generic sequence described above (SEQ ID NO.4, 5, 6, 7, or 10) such that it is capable of inducing tissue formation when properly folded and implanted in a mammal
  • the synthetic protein can induce bone formation from osteoblasts when implanted in a favorable environment, or it can promote cartilage formation when implanted in an avascular locus or when co-administered with an inhibitor of full bone development
  • the synthetic morphogenic protein of this invention comprises a sequence sufficiently duplicative of a partial or complete sequence of a COP, e.g , COP5 (SEQ ID NO 11) or COP7 (SEQ ID NO.12) Biosynthetic COP sequences are believed to dimerize during refolding and appear not to be active when reduced Both homodimeric and heterodimeric COPs are contemplated in this invention.
  • this synthetic protein is less than about 200 amino acids
  • osteogenic proteins may be used in concert with a hormone/receptor pair and tested using in vitro, ex vivo or in vivo bioassays for progenitor cell induction and tissue regeneration
  • the proteins in conjunction with the hormone/receptor pairs of this invention are envisioned to be useful for the repair and regeneration of bone, cartilage, tendon, ligament, neural and potentially other types of tissue Homologous Proteins Having Morphogenic Activity
  • the morphogenic proteins useful in this invention may be produced by recombinant expression of DNA sequences isolated based on homology with the osteogenic COP consensus sequences described above Synthetic COP DNA sequences may be used as probes to retrieve related DNA sequences from a variety of species (see, e.g , Oppermann et al, U.S Patent Nos.
  • Morphogenic proteins encoded by a gene that hybridizes with a COP sequence probe are assembled into two subunits disulfide-bonded to produce a heterodimer or homodimer capable of inducing tissue formation when implanted into a mammal
  • BMP-2 and BMP-4 have been shown to have cross-species osteogenic activity as homodimers and as heterodimers assembled with OP-1 subunits
  • Morphogenic protein-encoding genes that hybridize to synthetic COP sequence probes include genes encoding Vgl, inhibin, DPP, OP-1, BMP-2 and BMP-4 Vgl is a known Xenopus laevis morphogenic protein involved in early embryonic patterning Inhibin is another developmental gene that is a member of the BMP family of proteins from Xenopus laevis.
  • DPP is an amino acid sequence encoded by a Drosophila gene responsible for development of the dorso-ventral pattern
  • OP-1, BMP-2 and BMP-4 are osteogenic proteins that can induce cartilage, bone and neural tissue formation
  • a morphogenic protein may comprise a polypeptide encoded by a nucleic acid that hybridizes under stringent conditions to an "OPS" nucleic acid probe (Oppermann et al , U S Patent No 5,354,557) "OPS" - standing for OP-1 "short” - refers to the portion of the human OP-1 protein defining the conserved 6 cysteine skeleton in the C-terminal active region (97 amino acids, SEQ ID NO 2, residues 335-431)
  • stringent hybridization condition hybridization in 4X SSC at 65°C (or 10°C higher than the calculated melting temperature for a hybrid between the probe and a nucleic acid sequence containing no mis-matched base pairs), followed by washing in 0 IX SSC at the hybridization temperature
  • Another stringent hybridization condition is hybridization in 50% formamide, 4X SSC at 42°C
  • genes, or isolate genes from cDNA or genomic libraries that encode ammo acid sequences having morphogenic activity can be expressed in prokaryotic or eukaryotic host cells to produce large quantities of active osteogenic or otherwise morphogenic proteins
  • the recombinant proteins may be in native, truncated, mutant, fusion, or other active forms capable of inducing formation of bone, cartilage, or other types of tissue, as demonstrated by in vitro and ex vivo bioassays and in vivo implantation in mammals, including humans Hormones and Receptors Thereof
  • a hormone/receptor pair of this invention is capable of stimulating the ability of a morphogenic protein to induce tissue formation from a progenitor cell
  • the tissue inductive activity of a morphogenic protein in a mammal is improved by co-administering effective amounts of a hormone and a soluble receptor thereof
  • the morphogenic protein and the hormone/receptor pair are administered sequentially It has been found that the synergism between a morphogenic protein and a hormone/receptor pair is preserved even if the morphogenic protein is administered 4 to 8 hours before the hormone/receptor pair
  • the morphogenic protein, the hormone, and the hormone receptor can also be administered separately
  • One or more hormone/receptor pairs can be selected for use in concert with one or more morphogenic proteins according to the desired tissue type to be induced and the site at which the treatment will be administered
  • the particular choice of a morphogenic protein(s)/hormone(s)/receptor(s) combination and the relative concentrations at which they are combined may be
  • Hormones useful in this invention include, but are not limited to, interleukins 1 throughl ⁇ , fibroblast growth factor, vascular endothelial growth factor, platelet-derived growth factor, TGF- ⁇ , and prostaglandins (e g , El and E2) It may be preferred that the target cell has a cell-surface receptor for the hormone
  • the hormones can also be morphogenic proteins such as GDFs, as a result, the composition of this invention will contain two morphogenic proteins and a soluble receptor of one of these proteins
  • One preferred hormone/receptor pair of this invention is IL-6/sIL-6R IL-6 is a member of a subfamily of multifunctional hormones It appears to play a role in both bone formation and bone resorption by affecting mitogenesis of target cells and regulating the synthesis of other local factors Clinical studies show that IL-6 is involved in a variety of diseases, such as fibrous dysplasia, osteopenia, osteoporosis and Paget's disease Recombinant full length human
  • coli can be obtained from Sigma (St Louis, MI) and Promega (Madison, WI) Recombinant sIL-6R produced from baculovirus and containing the entire extracellular domain (residues 1-339, 38 kD) of human IL-6R can be obtained from Sigma and R&D Systems (Minneapolis, MN) See also Examples 1 and 2, infra Active allelic, species or other variants of these IL-6 and sIL-6 products can also be used
  • the hormone or the hormone receptor of this invention can be associated with an agent that is capable of increasing the hormone's or receptor's bio-activity, e g , synthesis, half-life, bio-availability, and reactivity with other bio-molecules such as binding proteins and receptors
  • agents may contain carrier molecules such as proteins and lipids
  • the hormone and hormone receptor are present in amounts capable of synergistically stimulating the tissue inductive activity of the morphogenic protein in a mammal
  • the relative concentrations of morphogenic protein, hormone and hormone receptor that optimally induce tissue formation may be determined empirically by the skilled practitioner using the procedures described herein Progenitor Cells
  • the progenitor cell that is induced to proliferate and/or differentiate by the morphogenic protein of this invention is preferably a mammalian cell.
  • progenitor cells are mammalian chondroblasts, osteoblasts and neuroblasts, all earlier developmental precursors thereof, and all cells that develop therefrom (e.g , pre- chondroblasts and chondrocytes)
  • the progenitor cell may be induced to form one or more tissue types such as endochondral or intramembranous bone, cartilage, tendon/ligament-like tissue, neural tissue and kidney tissue
  • tissue types such as endochondral or intramembranous bone, cartilage, tendon/ligament-like tissue, neural tissue and kidney tissue
  • the specific morphogenic activity exhibited by a morphogenic protein will depend in part on the type of the progenitor cell as well as the treatment site These variables may be tested empirically Morphogenic proteins are highly conserved throughout evolution, and non- mammalian progenitor cells are likely to be stimulated by same- or cross-species morphogenic proteins and hormone/receptor combinations It is thus envisioned that where schemes are available for implanting
  • a useful in vitro assay may monitor a nucleic acid or protein marker whose expression is known to correlate with the associated cell differentiation pathway See, e g , Examples 3 and 4 of United States Patent No 5,854,207, Lee et al.; and Examples 1 and 2, infra
  • OP-1 is known to have osteogenic and neurogenic activity
  • an in vitro assay that examines the expression of a molecular marker, e.g , an osteogenic- or a neurogenic-associated marker, in appropriate progenitor cells.
  • a molecular marker e.g , an osteogenic- or a neurogenic-associated marker
  • One useful assay for testing potential hormone/receptor pairs with OP-1 for osteogenic activity is the alkaline phosphatase ("AP") enzymatic assay.
  • AP is an osteoblast differentiation marker in primary osteoblastic fetal rat calvarial (“FRC”) cells.
  • the OP-1- stimulated AP activity results from increased steady-state AP mRNA levels
  • Other useful protein markers for monitoring osteogenic activity of a composition include, but are not limited to, type I collagen, osteocalcin, osteopontin, bone sialoprotein and PTH-dependent cAMP levels
  • An AP assay is performed generally as follows First, a hormone/receptor pair is identified by picking various concentrations and ratios of the hormone and hormone receptor and testing them in the absence and presence of a morphogenic protein Second, the amounts of hormone and hormone receptor required to achieve optimal, preferably synergistic, tissue induction in concert with the morphogenic protein is determined by generating dose response curves
  • additional hormone/receptor pairs that further stimulate or otherwise alter the morphogenic activity induced by a morphogenic protein and a first hormone/receptor pair may be identified and a new multi-factor dose response curve generated See, e g , Example 5 of United States Patent 5,854,207 Bone Induction Assays
  • compositions and devices of this invention can also be evaluated with ex vivo or in vivo bioassays
  • a rat bioassay for bone induction may be used to monitor osteogenic activity of osteogenic proteins in concert with one or more hormone/receptor pairs See, e g , Sampath et al , Proc. Natl Acad. Sci. USA, 80, pp 6591-95 (1983), and United States Patent No 5,854,207, Example 7 Rat bioassays are useful as the first step in moving from in vitro studies to in vivo studies
  • Assays for monitoring tendon and ligament-like tissue formation induced by morphogenic proteins are known in the art See, e g , Celeste et al , WO 95/16035, hereby incorporated by reference Such assays can be used to identify hormone/receptor pairs that stimulate tendon/ligament-hke tissue formation by BMP- 12, BMP- 13 or other morphogenic proteins in a particular treatment site The assays may also be used to optimize concentrations and treatment schedules for therapeutic tissue repair regiments These assays may be used to test various combinations of morphogenic protein and hormone/receptor combinations, and to produce an in vivo dose response curve for determining the effective relative concentrations of morphogenic proteins and hormones/receptors Neural Assays
  • the osteogenic proteins BMP-4 and BMP-7 (OP-1) can induce ventral neural plate explants to undergo differentiation into dorsal neural cell fates (Liem et al , Cell, 82, pp 969-79 (1995)) Molecular markers of dor
  • compositions of this invention contain at least one (e g , at least 2, 3 or 5) morphogenic protein, and at least one (e.g , at least 2 or 3) hormone/receptor pair These compositions are capable of inducing tissue formation when administered, e g , implanted, into a patient The compositions will be administered at an effective dose to induce the particular type of tissue at the desired treatment site
  • Determination of a preferred pharmaceutical formulation and a therapeutically efficient dose regiment for a given application is well within the skill of the art Factors that need to be considered include, for example, the administration mode, the condition and weight of the patient, the extent of desired treatment and the tolerance of the patient for the treatment
  • Doses expected to be suitable starting points for optimizing treatment regiments are based on the results of m vitro assays, and ex vivo or in vivo assays Based on the results of such assays, a range of suitable morphogenic protein and hormone/receptor concentration ratios can be selected to test at a treatment site in animals and then in humans
  • compositions of this invention may be in a variety of forms These include, for example, solid, semi-solid and liquid forms such as tablets, pills, powders, liquids, suspensions, suppositories, gels, pastes, and other injectable and infusible solutions
  • solid, semi-solid and liquid forms such as tablets, pills, powders, liquids, suspensions, suppositories, gels, pastes, and other injectable and infusible solutions
  • Modes of administration may include oral, parenteral, subcutaneous, intravenous, intralesional or topical administration
  • the pharmaceutical compositions of this invention will be administered in the vicinity of or at the treatment site in need of tissue regeneration or repair
  • compositions of this invention may, for example, be placed into sterile, isotonic formulations with or without co-factors which stimulate uptake or stability
  • the compositions may contain a formulation buffer comprising 5 0 mg/ml citric acid monohydrate, 2 7 mg/ml trisodium citrate, 41 mg/ml mannitol, 1 mg/ml glycine and 1 mg/ml polysorbate 20
  • This solution can be lyophilized, stored under refrigeration and reconstituted prior to administration with sterile Water-For-Injection (USP).
  • compositions may also include pharmaceutically acceptable carriers well known in the art See, for example, Remington's Pharmaceutical Sciences, 16th Edition, 1980, Mac Publishing Company
  • pharmaceutically acceptable carriers may include other medicinal agents, carriers, genetic carriers, adjuvants, and excipients such as human serum albumin or plasma preparations
  • the compositions may be in the form of a unit dose and will usually be administered as a dose regiment that depends on the particular tissue treatment
  • compositions of this invention may also be administered in form of a morphogenic device using, for example, microspheres, liposomes, other micro- particulate delivery systems or sustained release formulations placed in, near, or otherwise in communication with affected tissues or the bloodstream bathing those tissues
  • Liposomes containing the polypeptide mixtures of this invention can be prepared by well-known methods See, e g DE 3,218, 121 , Epstein et al , Proc. Natl. Acad. Sci. U.S.A., 82, pp 3688-92 (1985), Hwang et al , Proc. Natl. Acad. Set.
  • the liposomes are of the small (about 200-800 Angstroms) unilamellar type in which the lipid content is greater than about 30 mol % cholesterol The proportion of cholesterol is selected to control the optimal release rate of the polypeptides of interest
  • polypeptide mixtures of this invention may also be attached to liposomes containing other biologically active molecules to modulate the rate and characteristics of tissue induction
  • Such attachment may be accomplished by cross-linking agents such as heterobifunctional cross-linking agents that have been widely used to couple toxins or chemotherapeutic agents to antibodies for targeted delivery
  • Conjugation to liposomes can also be accomplished using the carbohydrate-directed cross-linking reagent 4-(4-maleimidophenyl) butyric acid hydrazide See, e g , Duzgunes et al . J. Cell. Biochem bst., Suppl 16E 77 Q992) Morphogenic Devices
  • compositions of this invention can additionally contain an implantable, biocompatible carrier Such compositions are also called morphogenic devices
  • the carrier functions as a sustained release delivery system for the therapeutic proteins and protects the proteins from non-specific proteolysis
  • the carrier may be biodegradable in vivo
  • a sustained release carrier may contain semipermeable polymer matrices in the form of shaped articles, e g , suppositories or capsules
  • Such a carrier can be made of polylactides (U S Patent No 3,773,319, EP 58,481), copolymers of L-glutamic acid and ethyl-L-glutamate (Sidman et al , Bwpolymers, 22, pp 547-56 (1985)), poly(2- hydroxyethyl-methacrylate) or ethylene vinyl acetate (Langer et al , J. Biomed. Mater. Res., 15, pp 167-277 (1981), Langer, Chem. Tech., 12,
  • the carrier may also serve as a temporary scaffold and substratum for recruitment of migratory progenitor cells and their subsequent anchoring and proliferation, until replaced by new bone or other appropriate tissue
  • the carrier may contain a biocompatible matrix made up of particles or otherwise having the desired porosity or microtexture The pores will permit migration, anchoring, differentiation and proliferation of the relevant progenitor cells
  • the particle size may be within the range of 70 ⁇ m-850 ⁇ m, e g , 70 ⁇ m-420 ⁇ m or 150 ⁇ m-420 ⁇ m
  • a particulate matrix may be fabricated by close packing particulate materials into a shape spanning the tissue defect to be treated Various matrices known in the art can be employed See, e g , U S Patent Nos 4,975,526, 5, 162, 1 14 and 5,171,574, and WO 91/18558, all of which are herein incorporated by reference
  • Useful matrix materials include but are not limited to collagen, celluloses, including carboxymethyl cellulose, homo- or co-poly
  • xenogenic bone powder matrices may be treated with proteases such as trypsin
  • the xenogenic matrices are treated with one or more fibril-modifying agents to increase the intraparticle intrusion volume (porosity) and surface area
  • Useful modifying agents include solvents such as dichloromethane, trichloroacetic acid, acetonitrile and acids such as trifluoroacetic acid and hydrogen fluoride
  • a preferred fibril-modifying agent is in the form of a heated aqueous medium, preferably an acidic aqueous medium having a pH less than about 4 5, most preferably having a pH between about 2 and 4, inclusive
  • the acidic aqueous medium can, for instance, be 0 1% ace
  • Xenogenic bone matrices can be used in a variety of clinical settings In addition to its use as a matrix for bone formation in various orthopedic, periodontal, and reconstructive procedures, the matrix also may be used as a sustained release carrier, or as a collagenous coating for orthopedic or general prosthetic implants
  • Demineralized guanidine-extracted xenogenic bovine bone contains a mixture of additional materials that may be fractionated further using standard biomolecular purification techniques
  • bone extracts can be fractionated by chromatography, and the various extract fractions corresponding to the chromatogram peaks can be added back together to an active matrix Doing so may remove inhibitors of bone or tissue-inductive activity, thereby improving matrix properties
  • the morphogenic devices of this invention may additionally contain other hormones and trophic agents
  • the devices may also contain antibiotics, chemotherapeutic agents, enzymes, enzyme inhibitors and other bioactive agents These ingredients may be adsorbed onto or dispersed within the carrier, and will be released over time at the implantation site as the carrier material is slowly absorbed General Consideration of Matrix Properties
  • Factors influencing the performance of a matrix include matrix geometry, particle size (if the matrix is made up of particles), the methodology for combining the matrix and morphogenic proteins, the degree of both intra- and inter-particle porosity, the presence of mineral, and the presence of surface charge
  • Particle size also influences the quantitative response of new bone, with sizes between 70 ⁇ m and 420 ⁇ m capable of eliciting the maximum response
  • contamination of the matrix with bone mineral may inhibit bone formation
  • Individual heavy metal concentrations in a bone matrix can be reduced to less than about 1 ppm by the methods described herein
  • the sequential cellular reactions at the interface of the bone matrix and an osteogenic protein implant are complex
  • the multi-step cascade includes binding of fibrin and fibronectin to the implanted matrix, migration and proliferation of mesenchymal cells, differentiation of the progenitor cells into chondroblasts, cartilage formation, cartilage calcification, vascular invasion, bone formation, remodeling, and bone marrow differentiation
  • a successful matrix is capable of accommodating each of these steps
  • the matrix may be shaped as desired in anticipation of surgery or shaped by the physician or technician during surgery It has been shown that new bone is formed essentially with the dimensions of the implanted device In the case where the matrix material is biodegradable in vivo, the matrix material is slowly absorbed by the body and is replaced by new bone in the shape of, or very nearly the shape of, the implant Thus, the matrix is preferably shaped to span a tissue defect and to take the desired form of the new tissue For example, in the case of bone repair of a non-union defect, it is desirable to use dimensions that span the non-union, and the new bone will eventually fill the defect
  • the matrix may be a shape-retaining solid made of loosely-adhered particulate material, e g , collagen Alternatively, the matrix may be a molded, porous solid, or an aggregation of close-packed particles held in place by surrounding tissue Masticated muscle or other tissue may also be used Large allogenic bone implants can act as a carrier for the matrix if their marrow cavities are cleaned and
  • the matrix may also take the form of a paste or a hydrogel "Hydrogel” refers to a three dimensional network of cross-linked hydrophihc polymers in the form of a gel
  • the gel is substantially composed of water, for instance, greater than 90% water
  • Hydrogel matrices can carry a net positive or net negative charge, or may be neutral
  • a typical net negative charged matrix is alginate
  • Hydrogels carrying a net positive charge are, for example, extracellular matrix components such as collagen and laminin
  • Examples of commercially available extracellular matrix components include MATRIGEL and VITROGENTM
  • Example of a net neutral hydrogel are highly cross-linked polyethylene oxide and polyvinyl alcohol
  • This invention also features an implantable prosthetic device comprising at least one morphogenic protein and at least one hormone/receptor pair at therapeutic amounts and ratios
  • the device can be used in conjunction with a composition containing the same or other morphogenic protein or hormone/receptor pair
  • the prosthetic device may be made from a material
  • compositions, devices and methods of this invention will permit a physician to treat a variety of tissue injuries, tissue degenerations, and other diseased tissue conditions
  • the compositions and devices can ameliorate or remedy these conditions by stimulating local tissue formation or regeneration
  • the devices of this invention may be implanted at the desired locus in a mammal such that the implant is accessible to the appropriate progenitor cells of this mammal
  • the devices may be used alone or in combination with other therapies for tissue repair and regeneration
  • the morphogenic devices of this invention may also be implanted in or surrounding a joint for use in cartilage and soft tissue repair, or in or surrounding nervous system-associated tissue for use in neural regeneration and repair
  • the tissue specificity of the particular morphogenic protein — or combination of morphogenic proteins with other biological factors - will determine the cell types or tissues that will be amenable to such treatments and can be selected by one skilled in the art
  • the ability to enhance morphogenic protein-induced tissue regeneration by co-administering a hormone/receptor pair according to the present invention is thus not believed to be limited to any particular cell-type or tissue
  • the osteogenic compositions and devices of this invention will permit the physician to obtain predictable bone, ligament and/or cartilage formation using less osteogenic protein to achieve at least about the same extent of bone or cartilage formation
  • the osteogenic compositions and devices of this invention may be used to treat more effectively the injuries, anomalies and disorders that have been described in the prior art of osteogenic devices These include, for example, forming local bone in fractures, non-union fractures, fusions and bony voids such as those created in tumor resections or those resulting from cysts, treating acquired and congenital craniofacial and other skeletal or dental anomalies (see e g , Glowacki et al , Lancet, 1, pp 959-63 (1981)), performing dental and periodontal reconstructions where lost bone replacement or bone augmentation is required such as in a jaw bone, and supplementing alveolar bone loss resulting from periodontal disease to delay or prevent tooth loss (see e g , NASAdsson et al , J. Penodontol,
  • An osteogenic device of this invention that comprises a matrix comprising allogenic bone may also be implanted at a site in need of bone replacement to accelerate allograft repair and incorporation in a mammal
  • Another potential clinical application of the improved osteogenic devices of this invention is in cartilage repair, for example, following joint injury or in the treatment of osteoarthritis
  • the ability to enhance the cartilage- inducing activity of morphogenic proteins by co-administering a hormone/receptor pair may permit faster or more extensive tissue repair and replacement using the same or lower levels of morphogenic proteins
  • the morphogenic compositions and devices of this invention will be useful in treating certain congenital diseases and developmental abnormalities of cartilage, bone and other tissues
  • homozygous OP-1 -deficient mice die within 24 hours after birth due to kidney failure (Luo et al , J.
  • heritable conditions including congenital bone diseases, for which use of the morphogenic compositions and devices of this invention will be useful include osteogenesis imperfecta, the Hurler and Marfan syndromes, and several disorders of epiphyseal and metaphyseal growth centers such as is presented in hypophosphatasia, a deficiency in alkaline phosphatase enzymatic activity
  • Inflammatory joint diseases may also benefit from the improved methods, compositions and devices of this invention
  • diseases include but are not limited to rheumatoid and pso ⁇ atic arthritis, bursitis, ulcerative colitis, regional enteritis, Whipple's disease, ankylosing spondy tis (also called Mane Strumpell or Bechterew's disease), and the so-called "collagen diseases” such as systemic lupus erythematosus (SLE), progressive systemic sclerosis (scleroderma), polymyositis (dermatomyositis), necrotizing vascuhtides, Sjogren's syndrome (sicca syndrome), rheumatic fever, amyloidosis, thrombotic thrombocytopenic purpura and relapsing polychondritis
  • Heritable disorders of connective tissue include Marfan' s syndrome, homocystinu ⁇ a, Ehlers-Danlos syndrome, osteogenesis imperfect
  • Example 1 Fig 1 shows the effects of 11-6, sIL-6R, and mixtures of recombinant human EL-6 and recombinant human sIL-6R ("IL-6/R"), respectively, on the OP-1 -induced AP activity in FRC cells
  • Confluent FRC cells were treated with the indicated agent(s) for 24 hrs
  • the concentrations of agent(s) used (ng/ml) are indicated in parentheses
  • IL-6/R the molar ratio of the two was maintained at about 1.2, and their respective amounts are indicated also in parentheses
  • Total AP activity was determined spectrophotometrically
  • Total cellular protein was determined by the Bradford Assay
  • IL-6 alone in the concentration range tested did not stimulate the basal AP activity SIL-6R alone stimulated the basal AP activity slightly, however, the stimulation did not seem to be sIL-6R dose-dependent
  • Fig 1 further demonstrates that IL-6 potentiates the OP-1 -induced AP activity in a dose-dependent manner A maximum of about 2-fold stimulation was observed (p ⁇ 0 05) SIL-6R also potentiated the OP-1 -induced AP activity in a dose-dependent manner A higher fold (about 3 5-fold, p ⁇ 0 02) of stimulation than observed with IL-6 was achieved
  • IL-6/R The effect of IL-6/R on the OP-1 -induced AP activity was also examined At the highest tested dose of IL-6 plus its soluble receptor (lOOng/ml IL-6 and 125ng/ml sIL-6R), the OP-1 -induced AP activity was synergistically enhanced by about 10-fold This enhancement was reproducible However, at the lower dose range, the IL-6/R combination did not appear to stimulate beyond what was achieved by either IL-6 or its receptor alone, on the contrary, the combination appeared to suppress AP activity
  • Fig. 2 shows that IL-6 alone enhanced OP-1 action in a mineralized bone nodule formation assay
  • FRC cells were grown in ⁇ MEN (supplemented with 5% FBS, 30 ⁇ g/ml gentamycin, 100 ⁇ g/ml ascorbic acid and 5 mM ⁇ -glycerolphosphate), and treated for various durations of time with (1) solvent vehicle, (2) 200ng/ml OP-1, or (3) 200ng/ml OP-1 plus IL-6 at various concentrations
  • the culture media were replenished with the same treatment agent(s) every three days
  • Progress of nodule formation was monitored every three days After a total of 15 days, cells were fixed with formalin and photographed
  • IL-6/R also enhanced OP-1 's ability to induce the formation of mineralized bone nodules
  • Example 3 To determine whether IL-6/R effects its synergy with OP-1 by directly stimulating OP-1 responsive cells or by increasing the number of OP-1 responsive cells, primary cultures of FRC were used as a model system, in which AP activity levels were used as a biochemical marker of OP-1 responsiveness Histochemical data showed that the number of AP positive cells in cultures treated with IL-6/R (40 ng/ml IL-6 and 50 ng/ml sIL-6R) and OP-1 (200 ng/ml) was similar to that in cultures treated with OP-1 alone (200 ng/ml) However, the AP activity level was higher in the former cultures than the latter cultures IL-6 alone (40 ng/ml) did not stimulate AP positive cells, and sIL-6R alone (50 ng/ml) or IL-6R (40 ng/ml IL-6 and 50 ng/ml sIL-6R) stimulated AP positive cells to a smaller extent, as compared to the combination of IL-6R and
  • BMPR-IA three BMP type I receptors
  • BMPR-IB BMPR-IB
  • ActR-I one BMP type II receptor
  • BMPR-II BMP type II receptor
  • RNA was isolated using the TRI reagent (Sigma) and loaded onto an AGAROSE GTG (FMC) gel containing formaldehyde Northern blots were prepared and probed with 32 P-labeled cDNA encoding for the various BMPRs These probes hybridized only to mRNA The radioactive bands were detected and quantified using a PHOSPHORIMAGER (Molecular Dynamics, Sunnyvale, CA) To normalize the band intensity of the BMPR bands, the blots were also probed with an oligonucleotide for the l8S rRNA
  • Example 5 The OP-1 protein used in Examples 1-5 was provided exogenously to the test cells To investigate whether the same IL-6/R synergistic effect would be observed when the OP-1 protein was expressed intracellularly in test cells, FRC cells were transfected with pW24, a plasmid carrying an OP-1 coding sequence under the control of the CMV promoter

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Abstract

La présente invention concerne des dispositifs et des méthodes permettant d'induire la formation tissulaire chez un mammifère, impliquant l'utilisation d'une protéine morphogénique, d'une hormone et d'un récepteur soluble de l'hormone. L'hormone et le récepteur correspondant sont utilisés pour améliorer l'activité inductive tissulaire de la protéine morphogénique.
EP00965478A 1999-09-27 2000-09-27 Compositions et methodes therapeutiques utilisant des proteines morphogeniques, des hormones et des recepteurs d'hormones Ceased EP1220909A2 (fr)

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WO2005111069A2 (fr) * 2004-05-11 2005-11-24 Stryker Corporation Proteines morphogeniques et facteurs de stimulation utilises en therapie genique
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CA2385887A1 (fr) 2001-04-05
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