Li, Rebecca H. (Bedford, MA, US)
Seeherman, Howard (Cambridge, MA, US)

Plaque It!

10/160607

08/19/2008

05/31/2002

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Wyeth (Madison, NJ, US)
Etex Corporation (Cambridge, MA, US)

424/717

424/494, 424/486, 514/2, 424/484, 424/602

A61K33/42; A61K9/14; A61K9/50; A61K33/00; A61K38/00

514/50

2465357	Therapeutic sponge and method of making	March, 1949	Correll et al.
3955719	Conically walled syringe providing a progressively tighter piston fit	May, 1976	Pheulpin
4159358	Method of bonding a bioglass to metal	June, 1979	Hench et al.	427/318
4191747	Corrective agent for the covering and/or filling of bone defects, method for the preparation of same and method of using the same	March, 1980	Scheicher
4294753	Bone morphogenetic protein process	October, 1981	Urist
4394370	Bone graft material for osseous defects and method of making same	July, 1983	Jefferies
4399216	Processes for inserting DNA into eucaryotic cells and for producing proteinaceous materials	August, 1983	Axel et al.
4419446	Recombinant DNA process utilizing a papilloma virus DNA as a vector	December, 1983	Howley et al.
4434094	Partially purified osteogenic factor and process for preparing same from demineralized bone	February, 1984	Seyedin et al.
4441915	Diurethanes and herbicidal compositions containing the same	April, 1984	Arndt et al.
4455256	Bone morphogenetic protein	June, 1984	Urist
4468464	Biologically functional molecular chimeras	August, 1984	Cohen et al.
4472840	Method of inducing osseous formation by implanting bone graft material	September, 1984	Jefferies
4553542	Methods and apparatus for joining anatomical structures	November, 1985	Schenck et al.
4563350	Inductive collagen based bone repair preparations	January, 1986	Nathan et al.
4596574	Biodegradable porous ceramic delivery system for bone morphogenetic protein	June, 1986	Urist
4608199	Bone protein purification process	August, 1986	Caplan et al.
4619989	Bone morphogenetic protein composition	October, 1986	Urist
4627982	Partially purified bone-inducing factor	December, 1986	Seyedin et al.
4642120	Repair of cartilage and bones	February, 1987	Nevo et al.
4662884	Prostheses and methods for promoting nerve regeneration	May, 1987	Stensaas et al.
4681763	Composition for stimulating bone growth	July, 1987	Nathanson et al.
4703008	DNA sequences encoding erythropoietin	October, 1987	Lin
4727028	Recombinant DNA cloning vectors and the eukaryotic and prokaryotic transformants thereof	February, 1988	Santerre et al.
4737578	Human inhibin	April, 1988	Evans et al.
4758233	Cream applicator	July, 1988	Phillips et al.
4761471	Bone morphogenetic protein composition	August, 1988	Urist
4766067	Gene amplification	August, 1988	Biswas
4767628	Continuous release pharmaceutical compositions	August, 1988	Hutchinson
4769328	Expression of biologically active PDGF analogs in yeast	September, 1988	Murray et al.
4774228	Polypeptide cartilage-inducing factors found in bone used in tissue proliferation	September, 1988	Seyedin et al.
4774322	Polypeptide cartilage-inducing factors found in bone	September, 1988	Seyedin et al.
4795804	Bone morphogenetic agents	January, 1989	Urist
4798885	Compositions of hormonally active human and porcine inhibin containing an α chain and 62 chain	January, 1989	Mason et al.
4804744	Osteogenic factors	February, 1989	Sen
4810691	Polypeptide cartilage-inducing factors found in bone	March, 1989	Seyedin et al.
4828990	Method for purifying an interferon	May, 1989	Higashi et al.
4839215	Biocompatible particles and cloth-like article made therefrom	June, 1989	Starling et al.	428/131
4843063	Polypeptide cartilage-inducing factors found in bone	June, 1989	Seyedin et al.
4851521	Esters of hyaluronic acid	July, 1989	della Valle et al.
4868161	Method for promoting nerve regeneration	September, 1989	Robert
4877864	Osteoinductive factors	October, 1989	Wang et al.
4886747	Nucleic acid encoding TGF-β and its uses	December, 1989	Derynck et al.
4908204	Reversibly blocked plasmin, t-PA hybrid fibrinolytic enzymes and pharmaceutical compositions and anti-thrombotic use thereof	March, 1990	Robinson et al.
4920962	Splint-like element for use in end-to-end nerve suture	May, 1990	Proulx
4923805	FSH	May, 1990	Reddy et al.
4955892	Neural cell adhesion protein nerve prosthesis	September, 1990	Daniloff
4963146	Multi-layered, semi-permeable conduit for nerve regeneration	October, 1990	Li
4968590	Osteogenic proteins and polypeptides	November, 1990	Kuberasampath et al.
4992274	Tissue plasminogen activator A-chain/urokinase B-chain hybrid protein	February, 1991	Robinson et al.
5011486	Composite nerve guidance channels	April, 1991	Aebischer et al.
5011691	Osteogenic devices	April, 1991	Oppermann et al.
5013649	DNA sequences encoding osteoinductive products	May, 1991	Wang et al.	435/69.1
5019087	Nerve regeneration conduit	May, 1991	Nichols
5024841	Collagen wound healing matrices and process for their production	June, 1991	Chu et al.
5026381	Multi-layered, semi-permeable conduit for nerve regeneration comprised of type 1 collagen, its method of manufacture and a method of nerve regeneration using said conduit	June, 1991	Li
5041538	Mammalian follistatin	August, 1991	Ling et al.
5071834	Purified activin B composition	December, 1991	Burton et al.
5089396	Nucleic acid encoding β chain prodomains of inhibin and method for synthesizing polypeptides using such nucleic acid	February, 1992	Mason et al.
5102807	Inhibin isolated from ovarian follicular fluid	April, 1992	Burger et al.
5106626	Osteogenic factors	April, 1992	Parsons et al.
5106748	DNA sequences encoding 5 proteins	April, 1992	Wozney et al.
5108753	Osteogenic devices	April, 1992	Kuberasampath et al.
5108922	DNA sequences encoding BMP-1 products	April, 1992	Wang et al.
5116738	DNA sequences encoding	May, 1992	Wang et al.
5118667	Bone growth factors and inhibitors of bone resorption for promoting bone formation	June, 1992	Adams et al.
5124316	Method for periodontal regeneration	June, 1992	Antoniades et al.
5141905	DNA sequences encoding BMP-7 proteins	August, 1992	Rosen et al.
5147399	Method of treating nerve defects through use of a bioabsorbable surgical device	September, 1992	Dellon et al.
5166058	DNA sequences encoding the osteoinductive proteins	November, 1992	Wang et al.
5166190	Method for increasing fertility in males	November, 1992	Mather et al.
5166322	Cysteine added variants of interleukin-3 and chemical modifications thereof	November, 1992	Shaw et al.
5168050	Mammalian expression of the bone morphogenetic protein-2B using BMP2A/BMP2B fusion	December, 1992	Hammonds et al.
5171579	Formulations of blood clot-polymer matrix for delivery of osteogenic proteins	December, 1992	Ron et al.
5187086	Molecules with antibody combining sites that catalyze hydrolysis reactions through use of a charged hapten	February, 1993	Janda
5187263	Expression of biologically active PDGE analogs in eucaryotic cells	February, 1993	Murray et al.
5202120	Methods of reducing glial scar formation and promoting axon and blood vessel growth and/or regeneration through the use of activated immature astrocytes	April, 1993	Silver et al.
5206028	Dense collagen membrane matrices for medical uses	April, 1993	Li
5208219	Method for inducing bone growth	May, 1993	Ogawa et al.
5215893	Nucleic acid encoding the ba chain prodomains of inhibin and method for synthesizing polypeptides using such nucleic acid	June, 1993	Mason et al.
5216126	Receptor polypeptides and their production and uses	June, 1993	Cox et al.
5217867	Receptors: their identification, characterization, preparation and use	June, 1993	Evans et al.
5218090	EGF receptor truncates	June, 1993	Connors
5229495	Substantially pure receptor like TGF-β 1 binding molecules and uses thereof	July, 1993	Ichijo et al.
5256418	Collagen constructs	October, 1993	Kemp et al.
5258494	Osteogenic proteins	November, 1993	Oppermann et al.
5266683	Osteogenic proteins	November, 1993	Oppermann et al.
5278145	Method for protecting bone marrow against chemotherapeutic drugs using transforming growth factor beta 1	January, 1994	Keller et al.
5284756	Heterodimeric osteogenic factor	February, 1994	Grinna et al.
5286654	Detection and purification of activin polypeptide	February, 1994	Cox et al.
5290271	Surgical implant and method for controlled release of chemotherapeutic agents	March, 1994	Jernberg
5292802	Collagen-polymer tubes for use in vascular surgery	March, 1994	Rhee et al.
5306307	Spinal disk implant	April, 1994	Senter et al.
5308889	Dehydrated collagen-polymer strings	May, 1994	Rhee et al.
5324519	Biodegradable polymer composition	June, 1994	Dunn et al.
5324775	Biologically inert, biocompatible-polymer conjugates	June, 1994	Rhee et al.
5328955	Collagen-polymer conjugates	July, 1994	Rhee et al.
5352715	Injectable ceramic compositions and methods for their preparation and use	October, 1994	Wallace et al.
5354557	Osteogenic devices	October, 1994	Oppermann et al.
5356629	Composition for effecting bone repair	October, 1994	Sander et al.
5364839	Osteoinductive pharmaceutical formulations	November, 1994	Gerhart et al.
5366875	Methods for producing BMP-7 proteins	November, 1994	Wozney et al.
5399346	Gene therapy	March, 1995	Anderson et al.
5411941	Heterodimeric osteogenic factor	May, 1995	Grinna et al.
5413989	Method and activin compositions for inducing bone growth	May, 1995	Ogawa et al.
5420243	Biologically active TGF-β2 peptides	May, 1995	Ogawa et al.
5422340	TGF-βformulation for inducing bone growth	June, 1995	Ammann et al.
5447725	Methods for aiding periodontal tissue regeneration	September, 1995	Damani et al.
5455041	Method for inducing periodontal tissue regeneration	October, 1995	Genco et al.
5455329	DNA sequences coding for PTH variants, PTH variants, expression vector, bacterial host, use and therapeutic composition	October, 1995	Wingender et al.
5457047	DNA Sequences coding for PTH variants, PTH variants, expression vector, bacterial host, use and therapeutic composition	October, 1995	Wingender et al.
5457092	Methods of promoting bone growth in mammals comprising administration of modified parathyroid hormone	October, 1995	Schluter et al.
5459047	BMP-6 proteins	October, 1995	Wozney et al.
5464440	Porous implant with two sets of pores	November, 1995	Johansson
5492697	Biodegradable implant for fracture nonunions	February, 1996	Boyan et al.
5508263	Heterodimeric osteogenic factor	April, 1996	Grinna et al.
5516654	Production of recombinant bone-inducing proteins	May, 1996	Israel
5520923	Formulations for delivery of osteogenic proteins	May, 1996	Tjia et al.
5538892	Nucleic acids encoding a TGF-β type 1 receptor	July, 1996	Donahoe et al.
5543394	Bone morphogenetic protein 5(BMP-5) compositions	August, 1996	Wozney et al.
5545616	Method for predicting and/or preventing preterm labor	August, 1996	Woodruff
5547854	DNA encoding a receptor for Mullerian inhibitory substance, misr1, and corresponding vectors, cells, probes, and recombinant methods	August, 1996	Donahoe et al.
5556767	Polynucleotide encoding macrophage inflammatory protein γ	September, 1996	Rosen et al.
5618924	BMP-2 products	April, 1997	Wang et al.
5631142	Compositions comprising bone morphogenetic protein-2 (BMP-2)	May, 1997	Wang et al.
5635372	BMP-15 compositions	June, 1997	Celeste et al.
5635373	Bone morphogenic protein-5(BMP-5) and DNA encoding same	June, 1997	Wozney et al.
5637480	DNA molecules encoding bone morphogenetic protein-10	June, 1997	Celeste et al.
5639638	DNA molecules encoding bone morpogenetic protein-11	June, 1997	Wozney et al.
5645592	Use of hydrogels to fix bone replacements	July, 1997	Nicolais et al.
5648467	Natural killer cell stimulatory factor	July, 1997	Trinchieri et al.
5650176	Synthesis of reactive amorphous calcium phosphates	July, 1997	Lee et al.	424/602
5650494	Process for refolding recombinantly produced TGF-β-like proteins	July, 1997	Cerletti et al.
5658882	Methods of inducting formation of tendon and/or ligament tissue comprising administering BMP-12, BMP-13, and/or MP-52	August, 1997	Celeste et al.
5661007	Bone morphogenetic protein-9 compositions	August, 1997	Wozney et al.
5674292	Terminally sterilized osteogenic devices and preparation thereof	October, 1997	Tucker et al.
5676976	Synthesis of reactive amorphous calcium phosphates	October, 1997	Lee et al.	424/602
5688678	DNA encoding and methods for producing BMP-8 proteins	November, 1997	Hewick et al.
5693779	Production and use of anti-dorsalizing morphogenetic protein	December, 1997	Moos, Jr. et al.
5700664	Mammalian cytokine, IL-11	December, 1997	Yang et al.
5703043	Bone morphogenetic protein-10 (BMP-10) compositions	December, 1997	Celeste et al.
5728679	BMP-15 compositions	March, 1998	Celeste et al.
5750651	Cartilage and bone-inducing proteins	May, 1998	Oppermann et al.
5752974	Injectable or implantable biomaterials for filling or blocking lumens and voids of the body	May, 1998	Rhee et al.
5756457	Neural regeneration using human bone morphogenetic proteins	May, 1998	Wang et al.
5786217	Methods and compositions for the repair of articular cartilage defects in mammals	July, 1998	Tubo et al.
5789543	Vertebrate embryonic pattern-inducing proteins and uses related thereto	August, 1998	Ingham et al.	530/350
5813411	Method of deforming tissue with a swollen hydrogel	September, 1998	Van Bladel et al.
5827733	Growth differentiation factor-8 (GDF-8) and polynucleotides encoding same	October, 1998	Lee et al.
5846931	Compositions comprising bone morphogenic proteins and truncated parathyroid hormone related peptide and methods of inducing cartilage by administration of same	December, 1998	Hattersley et al.
5849880	Bone morphogenetic protein (BMP)--6	December, 1998	Wozney et al.
5866364	Recombinant bone morphogenetic protein heterodimers	February, 1999	Israel et al.
5932216	Antibodies to bone morphogenetic protein-10 (BMP-10)	August, 1999	Celeste et al.
5935594	Process and device for treating and healing a tissue deficiency	August, 1999	Ringeisen et al.
5936067	Macrophage inflammatory protein variants	August, 1999	Graham et al.
5939323	Hyaluronan based biodegradable scaffolds for tissue repair	August, 1999	Valentini et al.
5939388	Methods of administering BMP-5 compositions	August, 1999	Rosen et al.
5965403	Nucleic acids encoding bone morphogenic protein-16 (BMP-16)	October, 1999	Celeste et al.
5972368	Bone graft composites and spacers	October, 1999	McKay
5986058	Polynucleotide encoding growth differentiation factor-7 and protein encoded thereby	November, 1999	Lee et al.
6001352	Resurfacing cartilage defects with chondrocytes proliferated without differentiation using platelet-derived growth factor	December, 1999	Boyan et al.
6004937	Use of follistatin to modulate growth and differentiation factor 8 [GDF-8] and bone morphogenic protein 11 [BMP-11]	December, 1999	Wood et al.
6027919	BMP-12 and BMP-13 proteins and DNA encoding them	February, 2000	Celeste et al.
6034061	BMP-9 compositions	March, 2000	Rosen et al.
6034062	Bone morphogenetic protein (BMP)-9 compositions and their uses	March, 2000	Thies et al.
6077076	Bone augmentation for prosthetic implants and the like	June, 2000	Comfort
6132214	Medical implant	October, 2000	Suhonen et al.
6150328	BMP products	November, 2000	Wang et al.
6177406	BMP-3 products	January, 2001	Wang et al.
6187742	Method for healing and repair of connective tissue attachment	February, 2001	Wozney et al.
6190880	Recombinant bone morphogenetic protein heterodimers, compositions and methods of use	February, 2001	Israel et al.
6207813	BMP-6 proteins	March, 2001	Wozney et al.
6245889	BMP-4 products	June, 2001	Wang et al.
6284872	Tendon-inducing compositions	September, 2001	Celeste et al.
6287816	BMP-9 compositions	September, 2001	Rosen et al.
6291206	BMP receptor proteins	September, 2001	Wozney et al.
6331612	Bone morphogenic protein-16 (BMP-16) compositions	December, 2001	Celeste et al.
6340668	Neuronal uses of BMP-11	January, 2002	Celeste et al.
6432919	Bone morphogenetic protein-3 and compositions	August, 2002	Wang et al.
6437111	Bone morphogenetic protein-11 (BMP-11) compositions	August, 2002	Wozney et al.
6558925	Stem cell inhibitor	May, 2003	Graham et al.
6586388	Method of using recombinant osteogenic protein to repair bone or cartilage defects	July, 2003	Oppermann et al.
6593109	Recombinant bone morphogenetic protein heterodimers, compositions and methods of use	July, 2003	Israel et al.
6610513	Receptor proteins	August, 2003	Wozney et al.
6613744	BMP-6 proteins	September, 2003	Wozney et al.
6623934	Bone morphogenetic protein-16 (BMP-16)antibodies	September, 2003	Celeste et al.
6699471	Injectable hyaluronic acid derivative with pharmaceuticals/cells	March, 2004	Radice et al.
6719968	Tendon-inducing compositions	April, 2004	Celeste et al.
20020193883	Injectable porous bone graft materials	December, 2002	Wironen	623/23.56

EP0052510	May, 1982			Microencapsulation of water soluble polypeptides
EP0058481	August, 1982			Continuous release pharmaceutical compositions
EP0061840	October, 1982			Triazole compounds, a process for preparing them, their use as plant fungicides and fungicidal compositions containing them.
EP0121976	October, 1984			Partially purified osteogenic factor and process for preparing same from demineralized bone or an osteosarcoma.
EP0128041	December, 1984			Polypeptides exhibiting skeletal growth factor activity.
EP0155476	September, 1985			Production of polypeptides in insect cells.
EP0169016	January, 1986			Polypeptide cartilage-inducing factors found in bone
EP0177343	April, 1986			DNA, cell cultures and methods for the secretion of heterologous proteins and periplasmic protein recovery
EP0212474	March, 1987			BONE MORPHOGENETIC AGENTS
EP0222491	May, 1987			Nucleic acid encoding the alpha or beta chains of inhibin and method for synthesizing polypeptides using such nucleic acid
EP0241809	October, 1987			Synergistic association of amantadine and selegiline.
EP0313578	May, 1989			NOVEL OSTEOINDUCTIVE COMPOSITIONS.
EP0329239	August, 1989			Surgical aid endowed with osteotropic activity.
EP0394418	October, 1990			OSTEOGENIC FACTORS.
EP0401055	December, 1990			Bone Calcification factor.
EP0409472	January, 1991			Bone morphogenetic protein
EP0416578	March, 1991			Protein, DNA and use thereof.
EP0429570	June, 1991			OSTEOINDUCTIVE COMPOSITIONS.
EP0433225	June, 1991			Process for the production of biologically active protein (e.g. TGF).
EP0530804	March, 1993			Kits and compositions for the treatment and repair of defects or lesions in cartilage or bone
EP0531448	March, 1993			MAMMALIAN EXPRESSION OF THE BMP-2 FAMILY.
EP0336394	July, 1994			Receiving sheets for dye transfer type thermal printing.
EP0626451	November, 1994			Heterodimers of a TGF-beta superfamily.
EP0741187	November, 1996			Recombinant obese (Ob) proteins
EP0592562	November, 1999			BMP-9 COMPOSITIONS.
EP0536186	November, 2001			BONE AND CARTILAGE INDUCTIVE PROTEINS.
EP0688869	March, 2003			Novel osteoinductive compositions
EP0831884	July, 2003			METHODS AND COMPOSITIONS FOR HEALING AND REPAIR OF CONNECTIVE TISSUE ATTACHMENT
JP05123390	July, 2000			BONE FILLER
JP05277174	April, 2001			BIOIMPLANTATION MATERIAL
WO/1984/001106	March, 1984			REPAIR OF TISSUE IN ANIMALS
WO/1985/004173	September, 1985			BONE PROTEIN PURIFICATION PROCESS
WO/1986/000525	January, 1986			PROCESS FOR THE PREPARATION OF A PHARMACEUTICAL COMPOSITION INFLUENCING THE TISSUE METABOLISM AND HAVING A REGENERATING ACTION
WO/1986/000639	January, 1986			LYMPHOKINE PRODUCTION AND PURIFICATION
WO/1987/000528	January, 1987			INHIBIN AND METHOD OF PURIFYING SAME
WO/1988/000205	January, 1988			NOVEL OSTEOINDUCTIVE COMPOSITIONS
WO/1988/009787	October, 1989			NITROGEN CONTAINING ANTI-OXIDANT COMPOSITIONS
WO/1989/009788	October, 1989			BIOSYNTHETIC OSTEOGENIC PROTEINS AND OSTEOGENIC DEVICES CONTAINING THEM
WO/1989/010133	November, 1989			STEM CELL INHIBITORS
WO/1989/010409	November, 1989			BONE AND CARTILAGE INDUCTIVE COMPOSITIONS
WO/1990/003733	April, 1990			OSTEOGENIC FACTORS
WO/1990/011366	October, 1990			OSTEOINDUCTIVE COMPOSITIONS
WO/1991/002744	March, 1991			BONE-SPECIFIC PROTEIN
WO/1991/004274	April, 1991			METHOD FOR INHIBITING GROWTH OF STEM CELLS
WO/1991/005802	May, 1991			OSTEOGENIC DEVICES
WO/1991/010444	July, 1991			METHOD FOR INCREASING FERTILITY IN MALES
WO/1991/018047	November, 1991			MAMMALIAN EXPRESSION OF THE BMP-2 FAMILY
WO/1991/018098	November, 1991			BONE AND CARTILAGE INDUCTIVE PROTEINS
WO/1992/005198	April, 1992			EXPRESSION OF MACROPHAGE INDUCIBLE PROTEINS (MIPs) IN YEAST CELLS
WO/1992/005199	April, 1992			BMP-5 DERIVATIVES
WO/1992/007004	April, 1992			OSTEOGENIC PROTEIN
WO/1992/020793	November, 1992			CLONING AND RECOMBINANT PRODUCTION OF RECEPTOR(S) OF THE ACTIVIN/TGF-$g(b) SUPERFAMILY
WO/1992/022319	December, 1992			SUBSTANTIALLY PURE RECEPTOR LIKE TGF-beta1 BINDING MOLECULES AND USES THEREOF
WO/1993/000049	January, 1993			OSTEOGENIC FACTOR
WO/1993/000050	January, 1993			PHARMACEUTICAL FORMULATIONS OF OSTEOGENIC PROTEINS
WO/1993/000432	January, 1993			BMP-9 COMPOSITIONS
WO/1993/004692	March, 1993			MORPHOGEN-INDUCED MODULATION OF INFLAMMATORY RESPONSE
WO/1993/005751	April, 1993			OSTEOGENIC PROTEINS IN THE TREATMENT OF BONE DESEASES
WO/1993/006872	April, 1993			FORMULATIONS OF BLOOD CLOT-POLYMER MATRIX FOR DELIVERY OF OSTEOGENIC PROTEINS
WO/1993/009229	May, 1993			RECOMBINANT BONE MORPHOGENETIC PROTEIN HETERODIMERS, COMPOSITIONS AND METHODS OF USE
WO/1993/009802	May, 1993			TGF-BETA TO IMPROVE NEURAL OUTCOME
WO/1993/013206	July, 1993			STEM CELL INHIBITING PROTEINS
WO/1993/016099	August, 1993			DNA SEQUENCES ENCODING NOVEL GROWTH/DIFFERENTIATION FACTORS
WO/1993/019177	September, 1993			FOUR NOVEL RECEPTORS OF THE TGF-$g(b) RECEPTOR FAMILY
WO/1993/020858	October, 1993			BIOMATERIALS FOR BONE REPLACEMENTS
WO/1994/001557	January, 1994			BONE FORMATION-INDUCING PROTEIN
WO/1994/003200	February, 1994			MORPHOGEN-INDUCED NERVE REGENERATION AND REPAIR
WO/1994/006449	March, 1994			MORPHOGEN-INDUCED LIVER REGENERATION
WO/1994/011502	May, 1994			ACTIVIN RECEPTOR-LIKE KINASES, PROTEINS HAVING SERINE THREONINE KINASE DOMAINS AND THEIR USE
WO/1994/015949	July, 1994			GROWTH DIFFERENTIATION FACTOR-5
WO/1994/015965	July, 1994			GROWTH DIFFERENTIATION FACTOR-3
WO/1994/015966	July, 1994			GROWTH DIFFERENTIATION FACTOR-9
WO/1994/021681	September, 1994			GROWTH DIFFERENTIATION FACTOR-8
WO/1994/024285	October, 1994			MACROPHAGE INFLAMMATORY PROTEIN VARIANTS
WO/1994/026892	November, 1994			BMP-11 COMPOSITIONS
WO/1994/026893	November, 1994			BMP-10 COMPOSITIONS
WO/1995/001801	January, 1995			GROWTH DIFFERENTIATION FACTOR-6
WO/1995/001802	January, 1995			GROWTH DIFFERENTIATION FACTOR-7
WO/1995/005846	March, 1995			NEURAL REGENERATION USING HUMAN BONE MORPHOGENETIC PROTEINS
WO/1995/007982	March, 1995			ACTIVIN RECEPTORS-LIKE KINASE (ALK), BELONGING TO THE TGF RECEPTOR FAMILY AND/OR TO THE BMP RECEPTOR FAMILY
WO/2001/028602	April, 1995			FORMULATIONS OF HYALURONIC ACID FOR DELIVERY OF OSTEOGENIC PROTEINS
WO/1995/010539	April, 1995			GROWTH DIFFERENTIATION FACTOR-10
WO/1995/010611	April, 1995			METHOD OF INDUCING AND MAINTAINING NEURONAL CELLS
WO/1995/012664	May, 1995			ADAPTION OF MAMMALIAN CELL LINES TO HIGH CELL DENSITIES
WO/1995/015966	June, 1995			2-SPIRO(2'-SPIROCYCLOALKYL)CYCLOPROPYL CEPHALOSPORIN SULFONES AS ANTIINFLAMMATORY AND ANTIDEGENERATIVE AGENTS
WO/1995/018856	July, 1995			VERTEBRATE EMBRYONIC PATTERN-INDUCING HEDGEHOG-LIKE PROTEINS
WO/1995/033830	December, 1995			BMP-9 COMPOSITIONS
WO/1996/001845	January, 1996			GROWTH DIFFERENTIATION FACTOR-11
WO/1996/002559	February, 1996			GROWTH DIFFERENTIATION FACTOR-12
WO/1996/016668	June, 1996			NOVEL HEDGEHOG-DERIVED POLYPEPTIDES
WO/1996/018924	June, 1996			MICROSCOPE SYSTEM PROVIDED WITH OBSERVATION UNIT AND PHOTOGRAPHING UNIT
WO/1996/026710	September, 1996			COSMETIC AND/OR PHARMACEUTICAL PREPARATIONS
WO/1996/038570	December, 1996			NOVEL FUSION PROTEIN RECOVERY AND PURIFICATION METHODS
WO/1996/039170	December, 1996			CARTILAGE INDUCTION BY BONE MORPHOGENETIC PROTEINS
WO/1996/039203	December, 1996			MODIFIED OSTEOGENIC MATERIALS
WO/1996/040883	December, 1996			NOVEL FACTOR IX PURIFICATION METHODS
WO/1994/005800	March, 1997			DORSAL TISSUE AFFECTING FACTOR AND COMPOSITIONS
WO/1997/015321	May, 1997			PHARMACEUTICAL COMPOSITION CONTAINING AN ACTIVIN OR INHIBIN STIMULATOR
WO/1997/022308	June, 1997			MEDICAL IMPLANT
WO/1997/034626	September, 1997			METHODS FOR ENHANCING FUNCTIONAL RECOVERY FOLLOWING CENTRAL NERVOUS SYSTEM ISCHEMIA OR TRAUMA
WO/1997/040137	October, 1997			REGENERATION AND AUGMENTATION OF BONE USING MESENCHYMAL STEM CELLS
WO/1997/045532	December, 1997			HYALURONAN BASED BIODEGRADABLE SCAFFOLDS FOR TISSUE REPAIR
WO/1997/048275	December, 1997			ENDODERM, CARDIAC AND NEURAL INDUCING FACTORS
WO/1997/049412	December, 1997			AUTOCROSS-LINKED HYALURONIC ACID AND RELATED PHARMACEUTICAL COMPOSITIONS FOR THE TREATMENT OF ARTHROPATHIES
WO/1998/016641	April, 1998			ISOLATION AND METHOD OF USING TISSUE GROWTH-INDUCING FRZB PROTEIN
WO/1998/031788	July, 1998			INJECTABLE FORMULATIONS FOR TREATMENT OF OSTEOPOROTIC BONE
WO/1998/034951	August, 1998			A NEW CYTOKINE FAMILY AND USES THEREOF
WO/1998/040113	September, 1998			BONE PASTE
WO/1998/049296	November, 1998			HUMAN CERBERUS PROTEIN
WO/1999/001159	January, 1999			LINEAGE-RESTRICTED NEURONAL PRECURSORS
WO/1999/024070	May, 1999			ESTER DERIVATIVES OF HYALURONIC ACID WITH VISCOELASTIC PROPERTIES AND THEIR USE IN THE BIOMEDICAL AND HEALTHCARE FIELD
WO/1999/031120	June, 1999			NOVEL TGF-BETA PROTEIN PURIFICATION METHODS
WO/1999/037320	July, 1999			METHODS AND COMPOSITIONS FOR ENHANCING COGNITIVE FUNCTION USING MORPHOGENIC PROTEINS
WO/1999/038543	August, 1999			BONE PASTE SUBJECTED TO IRRADIATIVE AND THERMAL TREATMENT
WO/1999/045949	September, 1999			USE OF FOLLISTATIN TO MODULATE GDF-8 AND BMP-11
WO/1991/017777	November, 1999			INJECTABLE BIOACTIVE GLASS COMPOSITIONS AND METHODS FOR TISSUE RECONSTRUCTION
WO/2000/037124	June, 2000			INJECTABLE HYALURONIC ACID DERIVATIVE WITH PHARMACEUTICALS/CELLS
WO/2000/043781	July, 2000			GROWTH DIFFERENTIATION FACTOR INHIBITORS AND USES THEREFOR

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Henley III, Raymond J.

Finnegan, Henderson, Farabow, Garrett & Dunner LLP

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No. 60/296,818 filed on Jun. 8, 2001, the entire teachings of which are incorporated herein by reference.

We claim:

1. A composition for delivery of osteogenic proteins, which comprises an osteogenic protein as a first biologically active agent, a calcium phosphate material as a carrier, and an effective amount of a material that liberates gas upon dissolution in vivo.

2. The composition of claim 1, wherein the osteogenic protein is selected from the group consisting of members of the bone morphogenic protein (BMP) family.

3. The composition of claim 2, wherein the osteogenic protein is selected from the group consisting of BMP-2, BMP-4, BMP-5, BMP-6, BMP-7, BMP-10, BMP-12 and BMP-13, and combination thereof.

4. The composition of claim 2, wherein the osteogenic protein is BMP-2 or BMP-6, or a combination thereof.

5. The composition of claim 1, wherein the calcium phosphate material is selected from the group of calcium phosphates consisting of amorphous apatitic calcium phosphate, hydroxyapatite, tricalcium phosphate, and fluorapatite.

6. The composition of claim 1, wherein the calcium phosphate material is an amorphous apatitic calcium phosphate.

7. The composition of claim 1, wherein the calcium phosphate material is a poorly crystalline apatitic calcium phosphate.

8. The composition of claim 7, wherein the calcium phosphate has a calcium-to-phosphate ratio comparable to naturally occurring bone minerals.

9. The composition of claim 7, wherein the calcium phosphate material has a calcium-to-phosphate ratio of less than 1.50.

10. The composition of claim 7, wherein the calcium phosphate has a calcium-to-phosphate ratio of about 1.4.

11. The composition of claim 1, further comprising a supplementary material selected from the group consisting of pharmaceutically acceptable salts, polysaccharides, peptides, proteins, amino acids, synthetic polymers, natural polymers, and surfactants.

12. The composition of claim 1, further comprising a supplementary material selected from the group of solid structures consisting of sponges, meshes, films, fibers, gels, filaments, microparticles, and nanoparticles.

13. The composition of claim 1, further comprising a supplementary material selected from the group of bioerodible polymers consisting of collagen, glycogen, chitin, celluloses, starch, keratins, silk, nucleic acids, demineralized bone matrix, derivativized hyaluronic acid, polyanhydrides, polyorthoesters, polyglycolic acid, polylactic acid, and copolymers.

14. The composition of claim 1, further comprising a supplementary material selected from the group of polyesters consisting of α-hydroxycarboxylic acids, such as poly(L-lactide) (PLLA), poly(D,L-lactide) (PDLLA), polyglycolide (PGA), poly(lactide-co-glycolide) (PLGA), poly(D,L-lactide-co-trimethylene carbonate), and polyhydroxybutyrate (PHB), and polyanhydrides, and co-polymers.

15. The composition of claim 1, further comprising at least one supplementary material selected from the group consisting of SiO₂, Na₂O, CaO, P₂O₅, Al₂O₃ and CaF₂.

16. The composition of claim 1, further comprising a supplementary material selected from the group consisting of polysaccharides, peptides and fatty acids.

17. The composition of claim 1, further comprising a second active agent, wherein the second active agent is selected from the group consisting of Hedghog, Frazzled, Chordin, Noggin, Cerberus and Follistatin proteins.

18. A method of treating a bone defect in a mammal comprising administering to the site of the bone defect an effective amount of an osteogenic composition of claim 1.

19. The method of claim 18, wherein the osteogenic protein is selected from the group consisting of members of the bone morphogenic protein (BMP) family.

20. The method of claim 19, wherein the bone morphogenic protein is selected from the group consisting of BMP-2, BMP-4, BMP-5, BMP-6, BMP-7, BMP-10, BMP-12 and BMP-13 and combinations thereof.

21. The method of claim 19, wherein the bone morphogenic protein is BMP-2 or BMP-6, or a combination thereof.

22. The method of claim 18, wherein the calcium phosphate material is selected from the group of calcium phosphates consisting of amorphous apatitic calcium phosphate, hydroxyapatite, tricalcium phosphate, and fluorapatite.

23. The method of claim 18, wherein the calcium phosphate material is an amorphous apatitic calcium phosphate.

24. The method of claim 18, wherein the calcium phosphate material is a poorly crystalline apatitic calcium phosphate.

25. The method of claim 24, wherein the poorly crystalline apatitic calcium phosphate has a calcium-to-phosphate ratio comparable to naturally occurring bone minerals.

26. The method of claim 24, wherein the poorly crystalline apatitic calcium phosphate has a calcium-to-phosphate ratio of less than 1:1.50.

27. The method of claim 24, wherein the poorly crystalline apatitic calcium phosphate has a calcium-to-phosphate ratio of about 1:1.40.

28. The composition of claim 2, wherein the osteogenic protein is a bone morphogenic protein (BMP) heterodimer.

29. The composition of claim 3, wherein the osteogenic protein is a bone morphogenic protein (BMP) heterodimer.

30. The composition of claim 4, wherein the osteogenic protein is a bone morphogenic protein (BMP) heterodimer.

31. The method of claim 19, wherein the osteogenic protein is a bone morphogenic protein (BMP) heterodimer.

32. The method of claim 20, wherein the osteogenic protein is a bone morphogenic protein (BMP) heterodimer.

33. The method of claim 21, wherein the osteogenic protein is a bone morphogenic protein (BMP) heterodimer.

34. A composition for delivery of osteogenic proteins, which comprises a bone morphogenic protein as a first biologically active agent, a calcium phosphate material as a carrier, and an effective amount of a gas that is dissolved under pressure, wherein the gas is selected from the group consisting of carbon dioxide, air, nitrogen, helium, oxygen, and argon, and wherein the gas is liberated upon exposure to physiological conditions.

35. A composition for delivery of osteogenic proteins, which comprises a bone morphogenic protein as a first biologically active agent, a calcium phosphate material as a carrier, and an effective amount of sodium bicarbonate.

36. The composition of claim 35, wherein the sodium bicarbonate is present at a concentration of between about 10 and about 40 percent (w/w).

37. The composition of claim 36, wherein the sodium bicarbonate is present at a concentration of about 20 percent (w/w).

38. A method of treating a bone defect in a mammal comprising administering to the site of the bone defect an effective amount of an osteogenic composition, wherein the osteogenic composition comprises a bone morphogenetic protein, a calcium phosphate material, and sodium bicarbonate.

39. The method of claim 38, wherein the sodium bicarbonate is added at a concentration of between about 10 and about 40 percent (w/w).

40. The method of claim 38, wherein the osteogenic composition further comprises a supplementary material selected from the group consisting of pharmaceutically acceptable salts, polysaccharides, peptides, proteins, amino acids, synthetic polymers, natural polymers, and surfactants.

41. The method of claim 38, wherein the osteogenic composition further comprises a supplementary material selected from the group of polyesters consisting of α-hydroxycarboxylic acids, such as poly(L-lactide) (PLLA), poly(D,L-lactide) (PDLLA), polyglycolide (PGA), poly(lactide-co-glycolide (PLGA), poly(D,L-lactide-co-trimethylene carbonate), polyhydroxybutyrate (PHB), polyanhydrides, and co-polymers thereof.

42. The method of claim 38, wherein the osteogenic composition further comprises at least one supplementary material selected from the group consisting of SiO₂, Na₂O, CaO, P₂O₅, Al₂O₃ and CaF₂.

FIELD OF THE INVENTION

This invention relates to composite materials containing calcium phosphate useful as delivery vehicles for osteoinductive proteins. The invention further relates to biocompatible, osteoinductive composites that can be used for bone regeneration and osseous augmentation, as well as for tissue repair and reinforcement in bone fractures, dental implants, bone implants and prostheses and the like.

BACKGROUND OF THE INVENTION

Much research in the area of biopharmaceutics is directed toward the development of effective implantable vehicles for drug delivery and other surgical applications. Such vehicles must be biocompatible and also must be capable of protecting the activity of any biologically active agent they are intended to deliver. Many biologically active agents are labile and easily lose activity when they are incorporated into a delivery material. Preservation of protein activity has posed particularly difficult problems.

In the drug delivery arena, calcium phosphate ceramics have been studied as potential delivery vehicles due to their well known biocompatibility and their affinity for protein reagents (see, e.g., Ijntema et al, Int. J. Pharm. 112:215 (1994); Itokazu et al., J. Orth. Res. 10:440 (1992); Shinto et al., J. Bone Joint Surg. 74-B:600 (1992); and Uchida et al., J. Orth. Res. 10:440 (1992)). Most of these materials have been in the form of prefabricated, sintered hydroxyapatite in either granule or block forms. These preparations have several drawbacks, including a limited ability to conform to skeletal defects, particularly in the case of blocks; inadequate structural integrity of granules (which do not bond together); and difficulty in modeling the implant to the shape of missing skeletal tissue with both blocks and granules. The block form of hydroxyapatite provides structural support, but among other complications, must be held in place by mechanical means, which greatly limits its use and its cosmetic results. Also, it is very difficult to saw a hydroxyapatite block into a shape that fits the patient's individual defect. The granular form produces cosmetically better results, but has a very limited structural stability and is difficult to contain during and after a surgical procedure. In general, all of these products are ceramics, produced by high temperature sintering, and are not individually crystalline, but rather have their crystal boundaries fused together. Most ceramic-type materials are in general functionally biologically non-absorbable (having an absorption rate generally not exceeding on the order of 1% per year).

A porous, non-resorbable material based on coral allows intergrowth with bone, but ultimately becomes only approximately 20% bone with the remaining 80% subsisting as scar tissue. HA RESORB® made by Osteogen is a form of absorbable hydroxyapatite, but is not a cement. It is granular and not adhesive. HA RESORB® is loosely rather than adhesively packed into place. For large uses, it is replaced by bone too quickly. In the dental materials market, HAPSET® is a composition of calcium phosphate granules and cementable plaster of Paris (calcium sulfate). This material is not truly a hydroxyapatite and contains too much calcium sulfate for most biological uses. The calcium sulfate component of such a composition is resorbable, but not the calcium phosphate granules.

At least one class of calcium phosphate compositions are precursors for the formation of hydroxyapatite and are biologically compatible, and have two unique properties that are not attainable in other calcium phosphate biomaterials: (1) self-hardening to form a mass with sufficient strength for many medical and dental applications, and (2) when implanted in bone, the material resorbs slowly and is completely replaced by new bone formation with no loss in the volume or integrity of the tissue that receives the implant. See U.S. Pat. Nos. Re. 33,221 and Re. 33,161 to Brown and Chow, which teach preparation of calcium phosphate remineralization compositions and of a finely crystalline, non-ceramic, gradually resorbable hydroxyapatite material based on the same calcium phosphate composition.

A virtually identical calcium phosphate system, which consists of tetracalcium phosphate (TTCP) and monocalcium phosphate (MCP) or its monohydrate form (MCPM) was described by Constantz et al. (U.S. Pat. Nos. 5,053,212 and 5,129,905). This system reportedly involves conversion of the MCP to dicalcium phosphate, which reacts with TTCP and forms hydroxyapatite (HA), the major mineral component of teeth and bone, as the end product.

Another type of calcium phosphate composition comprises an amorphous, apatitic calcium phosphate as a reactant, a promoter, and an aqueous liquid to form a hardening paste. See, e.g., U.S. Pat. Nos. 5,650,176; 5,676,976; 5,683,461; 6,027,742; and 6,117,456 to Lee et al. This system provides a bioactive ceramic material that is biocompatible, bioresorbable and workable for long periods of time at room temperature. The bioactive ceramic material may be formed at low temperatures, is readily formable and/or injectable, and yet can harden to high strength upon further reaction. The bioactive ceramic material contains poorly crystalline apatitic calcium phosphate solids with calcium-to-phosphate (Ca/P) ratios comparable to naturally occurring bone minerals and having stiffness and fracture roughness similar to natural bone. The bioactive ceramic composite material is strongly bioresorbable and its biosorbability and reactivity can be adjusted to meet the demands of the particular therapy and/or implant site. The material may be prepared as bone plates, bone screws and other fixtures and medical devices, including veterinarian applications, which are strongly bioresorbable and/or ossifying.

One of the goals of reconstructive surgery is to be able to replace damaged tissue with new tissue, using either a patient's own cells or growth enhancing proteins. For example, researchers have endeavored to develop cartilage regeneration systems in which isolated chondrocytes are injected into a damaged area in the context of a polymer scaffold (see, e.g., Atala et al., J. Urol. 150:747 (1993); Freed et al., J. Cell. Biochem. 51:257 (1993) and references cited therein). Similar seeded scaffold systems have been studied in the context of bone repair, where osteoblast cells are utilized in conjunction with polymeric or ceramic supports (see, e.g., Elgendy et al., Biomater. 14:263 (1993); Ishaug et al., J. Biomed. Mater. Res. 28:1445 (1994)). Of particular interest are osteoinductive materials such as bone morphogenetic proteins (e.g., recombinant human BMP-2), demineralized bone matrix; transforming growth factors (e.g., TGF-β); and various other organic species known to induce bone formation.

Three general types of calcium phosphate-based scaffold materials have been designed specifically for use with seeded compositions. One type of scaffold material consists of pre-formed calcium phosphate-based granules with the bioactive substance bound on the external surface. In general, large granules (ideally 100-1000 μm) are required to avoid eliciting inflammatory responses. However, such large pre-fabricated granules are not easily injectable through small gauge needles required for percutaneous injection. In addition, factors can only be admixed with preformed granules resulting in surface coating rather than the factor being embedded or dispersed throughout the matrix. Embedding the factor allows for a more controlled release of biomolecules as the matrix is resorbed. Pre-formed granules are typically difficult to handle and apply. Furthermore, most pre-formed hydroxyapatite granules are produced by a sintering process rendering them essentially non-resorbable.

A second type of scaffold material for seeded compositions consists of implantable porous hydroxyapatite or tricalcium phosphate blocks. Implantable porous blocks may be prepared with varying degrees of porosity, typically using a dry mixture of controlled particle size reactants. Other methods of promoting porosity include chemical or physical etching and leaching. Although they generally provide sufficient support, porous blocks have several significant drawbacks. First, like the pre-fabricated granules described above, block scaffolds do not have the osteoinductive substance embedded throughout the volume, and thus prevent controlled release of the active substance. Second, implantable blocks are not injectable, and thus require a more intrusive implantation procedure. Finally, and importantly, monolithic blocks may impede the rate of bone formation for clinical applications where an acceleration of healing is desired over the normal time course of healing. This delay may be due to slow resorption of the solid carrier and subsequent delayed release of the active substance. The presence of the monolithic matrix may also obstruct cell migration and infiltration to the fracture site. Assuming the block matrix contains interconnecting channels between the pores, new bone growth will be dictated by the pores and bounds of the scaffold walls, thus limiting new bone formation.

A third type of scaffold material involves calcium phosphate cements. Unlike the prefabricated granules and monolithic blocks, cements are readily injectable and can have the osteoinductive substance embedded throughout the volume. However, these cements tend to form monolithic aggregates that are inherently microporous. Although macroporous versions using biodegradable pore-formers have been described (see, e.g., PCT publication No. WO 98/16209, which is incorporated herein by reference), these cements form monolithic scaffolds which contain channels rather than microporous granules which, as discussed above, significantly restricts new bone growth.

Accordingly, despite substantial endeavors in this field, there remains a need for a drug delivery vehicle that is biocompatible, readily resorbable, and not detrimental to drug activity. Ideally, the vehicle should be injectable; malleable to enable injection or implantation into various sized fractures and defects; promote homogeneous distribution of bioactive materials throughout the matrix, thus permitting controlled release of the active substance; and, finally, form discrete macrogranules upon administration to the surgical or defective site. Granulation is desirable to facilitate cell migration and infiltration for secretion of extracellular bone matrix, and to provide access for vascularization. Granules also provide high surface area for enhanced resorption and release of active substance, as well as increased cell-matrix interaction. The present invention solves these needs, providing materials and compositions useful in drug delivery and in tissue repair.

SUMMARY OF THE INVENTION

In one embodiment, the invention provides a composition for delivery of osteogenic proteins, comprising a calcium phosphate material, an effective amount of an effervescent agent, and a biologically active agent. The calcium phosphate material may be an amorphous apatitic calcium phosphate, hydroxyapatite, tricalcium phosphate, or fluorapatite. In a preferred embodiment, the calcium phosphate material is an amorphous apatitic calcium phosphate, for example a poorly crystalline apatitic calcium phosphate. The poorly crystalline apatitic calcium phosphate may have a calcium-to-phosphate (Ca:P) ratio comparable to naturally occurring bone minerals. In preferred embodiments, the Ca:P ratio is less than 1.5, preferably about 1.4. The osteogenic protein may be a member of the bone morphogenic protein (BMP) family, including BMP-2, BMP-4, BMP-5, BMP-6, BMP-7, BMP-10, BMP-12 and BMP-13. In a preferred embodiment, the osteogenic protein is BMP-2 or BMP-6. The effervescent agent may be gas selected from the group consisting of carbon dioxide, air, nitrogen, helium, oxygen, and argon. In a preferred embodiment, the effervescent is sodium bicarbonate. The sodium bicarbonate may be present at a concentration of between about 10 and about 40 percent (w/w). The composition may further comprise one or more supplementary materials, such as pharmaceutically acceptable salts, polysaccharides, peptides, proteins, amino acids, synthetic polymers, natural polymers, and surfactants; solid structures, such as sponges, meshes, films, fibers, gels, filaments, microparticles, and nanoparticles; bioerodible polymers, such as collagen, glycogen, chitin, celluloses, starch, keratins, silk, nucleic acids, demineralized bone matrix, derivativized hyaluronic acid, polyanhydrides, polyorthoesters, polyglycolic acid, polylactic acid, and copolymers and derivates thereof; alpha-hydroxycarboxylic acids, such as poly(L-lactide) (PLLA), poly(D,L-lactide) (PDLLA), polyglycolide (PGA), poly(lactide-co-glycolide (PLGA), poly(D,L-lactide-co-trimethylene carbonate), and polyhydroxybutyrate (PHB), and polyanhydrides, and co-polymers and derivatives thereof; SiO ₂ , Na ₂ O, CaO, P ₂ O ₅ , Al ₂ O ₃ and CaF ₂ , and polysaccharides, peptides and fatty acids. The composition may further comprise a second active agent, such as a Hedghog, Frazzled, Chordin, Noggin, Cerberus and Follistatin protein.

In another aspect, the invention relates to method of treating a mammal having a bone defect comprising administering to the site of bone defect an effective amount of an osteogenic composition, wherein the osteogenic composition comprises a bone morphogenetic protein, a calcium phosphate material, and an effervescent agent. In a preferred embodiment, the effervescent agent is sodium bicarbonate.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to osteoinductive compositions adapted for use in the repair, regeneration and augmentation of bone tissue. The composition comprises a biocompatible and bioresorbable calcium phosphate material, an effervescent agent, and a biologically active agent. Upon hardening, the calcium phosphate material provides a resorbable scaffold for new bone growth. The effervescent agent prevents the calcium phosphate from forming a unitary monolithic structure by facilitating the formation of discrete macrogranules, which disperse during hardening of the calcium phosphate. The biologically active agent stimulates increased osteogenic activity of present or infiltrating progenitor or other cells. The osteoinductive compositions are useful for osseous augmentation and regeneration of bone tissue, for example in osteopenic bone, as well as for tissue repair and reinforcement in bone fractures, dental implants, bone implants and prostheses and the like.

As used herein, a “calcium phosphate material” means a synthetic bone substitute material comprising calcium phosphate as the primary component. Suitable calcium phosphate-based materials are well known in the art and include, without limitation, amorphous apatitic calcium phosphate, hydroxyapatite, tricalcium phosphate, and fluorapatite. In a preferred embodiment, the calcium phosphate material is a poorly crystalline apatitic calcium phosphate solid having a calcium-to-phosphate (Ca/P) ratio comparable to naturally occurring bone minerals. Such materials may be produced using a combination of amorphous, apatitic calcium phosphate as a reactant, a promoter, and an aqueous liquid to form a hardening paste. In an alternative embodiment, the calcium phosphate material is produced by solid-state acid-base reaction of crystalline calcium phosphate reactants to form crystalline hydroxyapatite solids.

“Effervescent agent” refers to a gaseous substance or a substance, which produces bubbling, foaming or liberation of a gas.

As used herein, “amorphous” means a material with significant amorphous character. Significant amorphous character contemplates greater than 75% amorphous content, preferably greater than 90% amorphous content, and is characterized by a broad, featureless X-ray diffraction pattern.

“Bioactive” refers to a material that induces hard tissue formation in and about the implant. When implanted in soft tissue, the bioactivity may also require the presence of a growth or trophic factor, or the seeding of the implant with a hard tissue forming cell type.

The term “biocompatible,” as used herein, means that the material does not elicit a substantial detrimental response in the host. There is always concern, when a foreign object is introduced into a living body, that the object will induce an immune reaction, such as an inflammatory response that will have negative effects on the host. For example, although hydroxyapatite is generally considered to be “biocompatible,” significant inflammation and tissue necrosis have been observed when crystalline hydroxyapatite microcarriers are inserted intramuscularly in animals (see, for example, IJntema et al., Int. J. Pharm. 112:215 (1994)).

“Bioresorbable” refers to the ability of a material to be resorbed in vivo. The resorption process involves elimination of the original implant materials through the action of body fluids, enzymes or cells. Resorbed calcium phosphate may, for example, be redeposited as bone mineral, or by being otherwise reutilized within the body, or excreted. “Strongly bioresorbable,” as that term is used herein, means that at least 80% of the total mass of material implanted intramuscularly or subcutaneously is resorbed within one year. In preferred embodiments, the material will be resorbed within nine months, six months, three months, and ideally one month.

An “effective amount” of an effervescent agent is an amount sufficient to effect the formation of macrogranules upon hardening, and will depend upon the calcium phosphate material being used. Generally, the amount of effervescent agent is added in a range of from about 1 to 90 percent by weight, preferably about 1 to 50 percent by weight, and more preferably about 10 to 40 percent by weight.

As used herein, a “macrogranule” means a granule or particle of between about 100 microns and 1 millimeter in diameter. The macrogranular material formed upon hardening of the inventive calcium-phosphate composition is biocompatible (i.e., the macrogranules are of sufficient size to avoid eliciting an inflammatory response) and macroporous, as described below.

As used herein, “macroporous” refers to a hardened calcium phosphate material having pores of sufficient diameter to permit cell migration and infiltration. In a preferred embodiment, the macroporous material formed in accordance with the present invention has a pore diameter of greater than 30 microns, more preferably between about 30 and 200 microns, and most preferably between about 50 and 100 microns in diameter. The macroporous material of the present invention facilitates cell migration and infiltration for secretion of extracellular bone matrix, as well as enhancing cell-matrix interactions.

An “effective amount” of a biologically active agent is an amount sufficient to stimulate increased osteogenic activity of present or infiltrating progenitor or other cells. The amount will depend upon the size and nature of the defect being treated, as well as the composition of the calcium phosphate material being employed. Generally, the amount of biologically active agent to be delivered is in a range of from about 0.1 to about 100 mg; preferably about 1 to about 100 mg; and most preferably about 10 to about 80 mg.

An “effective amount” of a supplemental material is an amount sufficient to impart the desired mechanical or chemical property to the composite.

“Hardening” refers to the process by which the malleable calcium phosphate composition is transformed into a hardened calcium phosphate material. The calcium phosphate material is considered to be “hardened” when it is a substantially non-formable solid. Such a hardened calcium phosphate material has minimal compressibility and tends to undergo plastic as opposed to elastic deformation.

“Poorly crystalline apatitic calcium phosphate,” “PCA calcium phosphate” and “PCA material,” as those terms are used herein, describe a synthetic poorly crystalline apatitic calcium phosphate. The poorly crystalline apatitic (PCA) material is not necessarily restricted to a single calcium phosphate phase provided it has the characteristic X-ray diffraction (XRD) and FTIR pattern. A PCA calcium phosphate has substantially the same XRD spectrum as bone. The spectrum is generally characterized by only two broad peaks in the region of 20-35° with one centered at 26° and the other centered at 32°, and by FTIR peaks at 563 cm ⁻¹ , 1034 cm ⁻¹ , 1638 cm ⁻¹ and 3432 cm ⁻¹ (±2 cm ⁻¹ ). Sharp shoulders are observed at 603 cm ⁻¹ and 875 cm ⁻¹ , with a doublet having maxima at 1422 cm ⁻¹ and 1457 cm ⁻¹ .

“Hydrated precursor,” as used herein, refers to the paste or putty formed by hydration of the dry PCA precursors in the presence of a limited amount of aqueous solution (i.e., less than approximately 1 mL aqueous solution/1 g precursor powder). The hydrated precursor may comprise both reactants and products, in various combinations, depending on the extent to which the conversion has progressed. Both the “injectable” and “formable” PCA precursor pastes described herein are hydrated precursors. Preferred “injectable” hydrated precursors have a consistency appropriate for delivery through an 18-gauge hypodermic needle.

The term “promoter,” as used herein, describes a material or treatment that promotes hardening of a hydrated precursor and may enhance the amorphous calcium phosphate (ACP) to PCA calcium phosphate conversion. Some promoters participate in the conversion and are incorporated into the PCA material; others, known as “passive” promoters, are not involved in the conversion.

“Reactive” is used herein to refer to the ability of a calcium phosphate, when mixed with liquid to form a hydrated precursor, to undergo conversion to the PCA material in the presence of a promoter in association with hardening of the precursor materials. Preferred ACPs are characterized by an ability to convert completely, an ability to convert quickly with hardening, an ability to undergo conversion with otherwise inert compounds and/or an ability to convert into a substantially homogeneous PCA material. Where the ACP is reacted with a second calcium phosphate, the “conversion” can encompass conversion of both the ACP and the second calcium phosphate. The degree of hardening and the kinetics of the hardening process are also important elements of reactivity. Some ACPs are more reactive than others. An ACP is considered “highly reactive” if it undergoes conversion and hardening to a PCA material in the presence of a weak promoter, such as dicalcium phosphate dihydrate (DCPD). Preferred highly reactive ACPs produce a hardened PCA material in the presence of weakly promoting DCPD and water at 37° C. in less than twelve hours, with hardening being substantially complete in about one to five hours, and ideally 10-30 minutes.

The Calcium Phosphate Material

Calcium phosphate component of the present invention may be any biocompatible, calcium phosphate material known in the art. The calcium phosphate material may be produced by any one of a variety of methods and using any suitable starting components. For example, the calcium phosphate material may be produced using a combination of amorphous, apatitic calcium phosphate as a reactant, a promoter, and an aqueous liquid to form a hardening paste. Alternatively, the calcium phosphate material may be produced by solid-state acid-base reaction of crystalline calcium phosphate reactants to form crystalline hydroxyapatite solids. Other methods of making calcium phosphate matrix materials are known in the art.

Poorly Crystalline Apatitic (PCA) Calcium Phosphate

In one embodiment, the calcium phosphate material is poorly crystalline apatitic (PCA) calcium phosphate. PCA material is described in application U.S. Ser. No. 08/650,764 and U.S. Pat. No. 5,650,176, both of which are hereby incorporated by reference in their entireties herein. The material is also described in a set of related applications, entitled “Delivery Vehicle,” “Conversion of Amorphous Calcium Phosphate to Form a Novel Bioceramic,” “Orthopedic and Dental Ceramic Implants,” and “Bioactive Ceramic Composites,” each of which was filed on Oct. 16, 1997 and assigned to ETEX Corporation (Cambridge, Mass.) and is incorporated herein by reference. In light of the breadth of disclosures in each of these related applications, the details of the PCA materials will not be belabored here. A summary of its characteristics will suffice.

The PCA material is characterized by its biological resorbability and its minimal crystallinity. Its crystalline character is substantially the same as natural bone. PCA material also is biocompatible and not detrimental to the host.

The PCA material may be implanted in a patient in a paste or putty form (i.e., as a hydrated precursor). Since the inventive reaction that produces the homogenous, macroporous calcium phosphate material can be initiated outside the body, and proceeds slowly at room temperature, the possibility that the material will “set up” prior to application to the surgical site and become unusable is minimized. The reaction accelerates significantly under physiological conditions (i.e., body temperature and pressure) and the material hardens in place. This feature is particularly useful in the surgical setting, where custom fitting of the device to the implant location is typically required. For example, the PCA paste containing the effervescent agent and biologically active agent may be applied to and used to fill a fracture site.

Alternatively, the PCA material may be pre-hardened outside the body, loaded with the desired biologically active agent and effervescent agent, and implanted at a later time. This approach is useful in those situations where custom shapes are not essential, and where production of large numbers of implants is desired.

Generally, the formation reaction of the present invention is completed after application to the surgical site. The material typically hardens in less than five hours, and substantially hardens in about one to five hours, under physiological conditions. Preferably, the material is substantially hardened within about 10-30 minutes. The consistency and formability of the PCA material, as well as the speed of the formation reaction, may be varied according to the therapeutic need by modifying a few simple parameters (see, e.g., U.S. Pat. No. 6,027,742 to Lee et al, which is incorporated by reference in its entirety herein).

The conversion reaction that produces the PCA material may be initiated by adding distilled water to a mixture of the dry precursor components to form a thick hydrated precursor in the form of a paste or putty. Other aqueous agents such as buffers, saline, serum or tissue culture medium may be used in place of distilled water. In other embodiments, sufficient water may be added to the precursor powders to form a paste, which, upon addition of the other invention components, is readily injectable with an 18 gauge needle. Most often, the resulting bioresorbable calcium phosphate material will be “calcium deficient,” with a calcium to phosphate ratio of less than 1.5 as compared to the ideal stoichiometric value of approximately 1.67 for hydroxyapatite.

Suitable PCA materials may be identified by combining the PCA precursors, hydrating with a limited amount of water (so that a paste or putty is formed), and allowing to harden into a PCA material. Desirable precursors are capable of hardening in a moist environment, at or around body temperature in less than 5 hours and preferably within 10-30 minutes. Components which harden in this way may then be placed intramuscularly or subcutaneously in a test animal and checked for biological resorbability. Desirable materials are those that, when implanted as a 1-5 g pellet, are at least 80% resorbed within one year. Preferably, the material can be fully resorbed. Generally, it is easier to test resorption of gram quantities of material in subcutaneous sites.

The PCA material may be formed in a reaction that employs at least one amorphous calcium phosphate (ACP) precursor, and preferably employs an activated or reactive ACP (see, e.g., PCT application No. WO 98/16209; Examples 1-4). In some instances, the reaction may employ only one precursor ACP, which is converted in a controlled fashion in part or whole to the PCA material. Also, a non-participating promoter may be employed to facilitate conversion of the activated ACP to the PCA material. In any event, reactions that can be initiated outside of the body, that can be carried out in a paste-like configuration, and that significantly accelerate at 37° C. leading to a hardened calcium phosphate product are greatly preferred.

The conversion of ACP to PCA material is promoted in the presence of water. Generally, the ACP is provided as a powder and combined with any other reactants (e.g., a second calcium phosphate), and is exposed to a limited amount of water, so that a past or putty is formed. The hydrated precursor then hardens, and the hardening is associated with formation of the PCA material. The conversion of ACP to PCA calcium phosphate proceeds in a controlled fashion as a paste or putty which hardens predictably and which has utility in dental, orthopedic, or other therapeutic applications.

When amorphous calcium phosphate is used as the sole precursor to produce a resorbable PCA material, it is important to control the natural tendency of the ACP to convert to highly crystalline hydroxyapatite. On the other hand, the time course of conversion should be fast enough to have surgical utility. One approach is to combine a precursor ACP containing an inhibitor of crystal formation (see, e.g., WO 98/16209; Example 1) with an ACP that does not contain an inhibitor of crystal formation (e.g., a promoter). The reactants may be mixed in a dry state, with the appropriate particulate size and an excess of the inhibitor-containing ACP. The reactants can then be hydrated by addition of water, followed by an elevation in temperature (e.g., as occurs following introduction into the body), to convert the reactants to the PCA material. Other methods of controlled conversion involve the use of catalysts.

Crystalline Hydroxyapatite

In a second embodiment, the calcium phosphate material is crystalline hydroxyapatite (HA). Crystalline HA is described, for example, in U.S. Pat. Nos. Re. 33,221 and Re. 33,161 to Brown and Chow, both of which are herein incorporated by reference. The Brown and Chow patents teach preparation of calcium phosphate remineralization compositions and of a finely crystalline, non-ceramic, gradually resorbable hydroxyapatite carrier material based on the same calcium phosphate composition. A similar calcium phosphate system, which consists of tetracalcium phosphate (TTCP) and monocalcium phosphate (MCP) or its monohydrate form (MCPM), is described by Constantz et al. in U.S. Pat. Nos. 5,053,212 and 5,129,905, both of which are incorporated herein by reference. In this embodiment, the calcium phosphate material is produced by solid-state acid-base reaction of crystalline calcium phosphate reactants to form crystalline hydroxyapatite solids.

Crystalline HA materials (commonly referred to as dahllite) may be prepared such that they are flowable, moldable, and capable of hardening in situ (see U.S. Pat. No. 5,962,028 to Constantz). These HA materials (commonly referred to as carbonated hydroxyapatite) can be formed by combining the wet and dry reactants to provide a substantially uniform mixture, shaping the mixture as appropriate, and allowing the mixture to harden. Alternatively, precursor reaction mixtures can be administered to the surgical site and hardened and/or shaped in situ. During hardening, the mixture crystallizes into a solid and essentially monolithic apatitic structure.

The reactants will generally consist of a phosphoric acid source substantially free of unbound water, an alkali earth metal, particularly calcium, source, optionally crystalline nuclei, particularly hydroxyapatite or calcium phosphate crystals, calcium carbonate, and a physiologically acceptable lubricant, such as water, which may have various solutes. The dry ingredients may be pre-prepared as a mixture and subsequently combined with the liquid ingredients under conditions where substantially uniform mixing occurs.

The phosphoric acid source may be any partially neutralized phosphoric acid, particularly up to and including complete neutralization of the first proton as in calcium phosphate monobasic. Alternatively or additionally, it can consist of orthophosphoric acid, possibly in a crystalline form, that is substantially free of uncombined water. Calcium sources will generally include counterions such as carbonate, phosphate or the like, particularly dual sources of calcium phosphate and phosphate such as tetracalcium phosphate or tricalcium phosphate.

The various dry components may be combined prior to the addition of the wet components. Mixing combines the ingredients and can be used to regulate the extent of the inter-ingredient reactions. Any or all of the dry ingredients may be added prior to the initiation of mixing or prior to the completion of mechanical mixing. After mixing, the mixture is allowed to anneal while remaining quiescent, followed by an extended period of time during which the mixture hardens.

The Effervescent Agent

The present invention provides a novel process for producing a calcium phosphate matrix or scaffold material which “self-granulates” and disperses into hardened macrogranules or macroparticles under physiological conditions (i.e., post-administration). The calcium phosphate material may be any biocompatible, calcium phosphate material known in the art, such as the PCA calcium phosphate and crystalline hydroxyapatite materials described above. Surprisingly, the present inventors have discovered that the addition of an effervescent agent to these calcium phosphate materials substantially alters the biological, chemical and mechanical properties of the material, thereby significantly enhancing its therapeutic utility. The effervescent agent of the present invention may be any pharmaceutically acceptable substance which produces bubbling or liberation of a gas at physiological temperatures and/or pressures.

All of the currently available methods for producing calcium phosphate materials for use with seeded compositions suffer from certain inherent drawbacks, including limited injectability due to granule formation during production or preparation for administration in the syringe. Pre-fabricated calcium phosphate granules, to which the bioactive substance adheres, must be large (ideally 100-1000 μm) to avoid eliciting inflammatory responses. However, such large pre-fabricated granules are not easily injectable through small gauge needles required for percutaneous injection. Moreover, these granules are typically difficult to handle and apply, and many are produced by a sintering process rendering them essentially non-resorbable. In addition, the active agent can only be admixed with preformed granules resulting in surface coating, rather than being evenly embedded or dispersed throughout the material. Dispersion allows for a more controlled release of biomolecules as the matrix is resorbed.

In an important aspect of the invention, the ease of use of the inventive bioceramic material in a surgical setting is significantly improved over other bone substitute composite materials known in the art. Specifically, an effervescent agent is added to the other components of the composition (e.g., calcium phosphate material and any supplementary materials) to cause gas foaming or bubbling under specific conditions (i.e., physiological temperatures and/or pressures). The bubbling or effervescence induces granulation and dispersion of the calcium phosphate material upon injection or implantation in vivo. As the hardening and/or cement reaction proceeds, granulation occurs simultaneously and the active agent (which may be admixed with the other components or added to the mixture just prior to administration) is homogeneously dispersed throughout the volume of the individual granules.

The effervescent agent is added in an appropriate amount to prevent the formation of a monolithic calcium phosphate mass. The effervescent agent reacts quickly and completely with a wide variety of calcium phosphates and other calcium- or phosphorus-bearing materials to provide a homogeneous injectable delivery vehicle. Depending upon the particular calcium phosphate material, the effervescent agent is selected to sufficiently interfere with the hardening or cementing process to allow the formation of relatively uniform granules, but not to the extent that it renders calcium phosphate cement “non-reactive.” The addition of the effervescent agent causes substantial granulation to occur only after injection or implantation in vivo. As a result, granulation does not occur during the preparation of the calcium phosphate material and/or formulation of the cement prior to injection or implantation. Granules formed in the presence of an effervescent agent are sufficiently large to prevent an inflammatory reaction (typically greater than 30 μm), yet small enough to provide a significant surface area to volume ratio. The large surface area to volume ratio enables rapid resorption of the calcium phosphate material as new bone is regenerated. The large surface area also facilitates release of the biologically active agent, while still retaining the agent at the surgical site for the appropriate length of time required for bone induction. In addition, the large surface area to volume ratio facilitates cell migration and infiltration into the matrix for secretion of extracellular bone