Human Bone Morphogenetic Protein -2

BMP-2

Contents

Introduction Naturally Occurrence Natural Pathway Recombinant Creation Functions-Natural Functions- Recombinant Similarities and Differences How rhBMP-2 works rhBMP-2 Production rhBMP-2 Carriers

Market Facts-Leg Market Facts-Dental Market Facts-Spinal ICBG Cost of Bone Fusion INFUSE/InductOs Benefits of rhBMP-2 rhBmp-2 to market Infuse Problems

Introduction BMP-2

Human Bone Morphogenic Protein 2 is one of twenty BMPs. stimulates the differentiation of mesenchymal cells into osteoblasts and chondrocytes. BMP-2 along with BMP-7 are osteogenic BMPs: they have been demonstrated to potently induce osteoblast differentiation in a variety of cell types. Important role in the development of bone and cartilage formation during embryogenesis and involved also in cardiac cell differentiation and epithelial to mesenchymal transition BMP2 was recently linked to osteoporosis via Linkage analysis in extended osteoporosis families in Iceland found 3 variants in the BMP2 gene. The association was seen with many definitions of an osteoporotic phenotype, including osteoporotic fractures and low BMD (Bone Mineral Density)

Structure of BMP-2

Human Bone Morphogenetic Protein-2

BMP-2 is located on chromosome 20, at 20p12.3 Size: 396 amino acids; 44702 Da Subunit: Homodimer; disulfide-linked. Interacts with GREM2 (By similarity) and SOSTDC1 Subcellular location: Secreted

Protein Sequence
1 mvagtrclla lllpqvllgg aaglvpelgr rkfaaassgr pssqpsdevl sefelrllsm 61 fglkqrptps rdavvppyml dlyrrhsgqp gspapdhrle raasrantvr sfhheeslee 121 lpetsgkttr rfffnlssip teefitsael qvfreqmqda lgnnssfhhr iniyeiikpa 181 tanskfpvtr lldtrlvnqn asrwesfdvt pavmrwtaqg hanhgfvvev ahleekqgvs 241 krhvrisrsl hqdehswsqi rpllvtfghd gkghplhkre krqakhkqrk rlkssckrhp 301 lyvdfsdvgw ndwivappgy hafychgecp fpladhlnst nhaivqtlvn svnskipkac 361 cvptelsais mlyldenekv vlknyqdmvv egcgcr

BMP-2 Pathway

Synthesized within the cell in precursor form, each molecule has a hydrophobic leader or secretory sequence with the mature portion of the protein at the carboxy terminus marked by a highly conserved, seven cysteine repeat. Each mature BMP begins as 2 monomers of 120 amino acids each, which undergo disulfide linkage dimerization to form either a homologous or a heterologous protein chain. In the case of BMP-2 and BMP-7, the specific structure was first identified by isolating the bovine protein from bone extracts. BMP-2 binds to its receptor (BMPR), which phosphorylates and activates the intracellular signaling molecules Smad 1 and Smad 5 leads to the expression of the transcription factor Cbfa1 (Runx2), Results in the expression of several proteins critical for bone formation

The signal transduction pathway for bone morphogenetic protein 2.. This in turn which results in the expression of several proteins critical for bone formation

TGF- Pathway

BMP2(Bone morphogenetic protein 2) belongs to the transforming growth factor-beta (TGFB) superfamily. The encoded protein acts as a disulfidelinked homodimer and induces bone and cartilage formation. Transforming growth factor beta is involved in many cellular processes in both the adult organism and the developing embryo. TGF superfamily ligands bind to a type II receptor, which recruits and phosphorylates a type I receptor. The type II receptor phosphorylates a cytoplasmic domain on the type I receptor , activating the type I receptor The type I receptor then phosphorylates receptor-regulated SMADs (R-SMADs) which can now bind the coSMAD SMAD4 R-SMAD/coSMAD complexes accumulate in the nucleus where they act as transcription factors and participate in the regulation of target gene expression.

Recombinant BMP-2 How and Why

Bone can be described as having three components:
1. A mineral component, 2. A collagenous matrix 3. A growth protein component. Using experimental models in rats, some authors decalcified and separated the bone tissue in a component that contains a collagenous matrix and other component that contains growth proteins. The protein component was reconstituted with inactive matrix and implanted ectopically. New bone was observed after ten days. This growth factor component was called bone morphogenetic protein (Urist et al., 1979; Sampath & Reddi). Isolating the BMPs in human corpses bone, only 0.1 g BMP per kilogram of bone can be obtained. Already the rhBMP-2 can be produced in abundance. This protein has non-antigenic and non-immunogenic properties, not having risk of human disease transference because it is a protein produced by bio-engineered methods

Some point mutations in the BMP gene

rhBMP2- Production

Recombinant Human Bone Morphogenetic protein-2 produced in E.Coli is a homodimeric, non-glycosylated, Polypeptide chain containing 115 amino acids and having a molecular mass of 26018 Dalton. BMP-2 is purified by proprietary chromatographic techniques. BMP-2 was lyophilized from a concentrated (1mg/ml) sterile solution containing 10mM sodium citrate pH=3.5. The hBMP-2 cDNA encoding the mature peptide of the BMP-2 protein was amplified from RNA extracted from human osteosarcoma U2-05 cells. DNA sequencing revealed a 321- base pair DNA fragment encoding C-terminal 107 amino acid of hBMP-2 protein. In the recombinant plasmid, pMX-BMP2, BMP-2 expression was under the direct control of a PlPr thermoinducible promoter. rhBMP-2 expression in the DH5QL host cells was induced in incubation @ 42C. SDS-PAGE showed the molecular weight of rhBMP-2 to be about 12 kDa and the expression level of rh-BMP more than 15% of the total bacterial proteins after six-hour induction at 42C

Functions- BMP-2

Induce ectopic bone formation Function as morphogenetic factors for many other tissues and organs. Have critical regulatory roles during early embryogenesis, such as neurogenesis, mesoderm formation Upregulates expression and function of voltage-gated K+ channels in human pulmonary artery smooth muscle cells. Inhibit proliferation and induce apoptosis in normal human PASMC, whereas dysfunctional BMP signaling and downregulated K(V) channels are involved in pulmonary vascular medial hypertrophy associated with pulmonary hypertension.

The bifurcated segmented rays of the stump (left) are fused (right) when regenerated. Fusion is due to the ectopic expression of the bone morphogenetic protein, bmp2b

Functions: rhBMP
Recruits bone-forming cells
(Mesenchymal stem cells differentiate into osteoblasts)

to the surgical area and converts local cells to bone and Promote bone growth in several areas of the body In the spine, the rhBMP-2 grows bone in the disc space to join or fuse the vertebrae to reduce back pain and stabilize the spine. In certain tibial fractures, rhBMP-2 has been shown to help heal broken bones. In of jaw bone resorption, rhBMP2 may be placed in the section or sections of the jaw bone that need to be built back up in preparation Radiographic analysis of new bone formation. Significant bone formation could be detected by X-ray film in animals injected with AAV-BMP2. (a) 3 for dental implants. 12

weeks postinjection of high-titer AAV-BMP2 (10 VP); (b) 8 weeks postinjection of high-titer AAV-BMP2 (1012 VP); (c) 3 weeks postinjection of high-titer AAV-BMP2 (1012 VP); (d) 8 weeks postinjection of high-titer AAVBMP2 (1012 VP); (e) 8 weeks postinjection of low-titer AAV-BMP2 (5X1011 VP); (f) 8 weeks postinjection of AAV-EGFP (1012 VP). Arrows indicate the regional newly formed bone tissue.

How it works
During surgery, rhBMP-2 is
soaked onto and binds with an absorbable collagen sponge (ACS) that is designed to resorb, or disappear, over time. As the sponge dissolves, the rhBMP-2 stimulates the cells to produce new bone. The rhBMP-2 also goes away once it has completed its task of initiating the normal bone healing process. The most efficient vectors for gene
delivery are viruses (Adeno-associated
virus (AAV) )

Click picture for video

rhBMP-2 carrier (absorbable collagen sponge )

VIDEO

Carriers for rhBMP-2

Delivery Material: Carrier for BMP needs to perform a threefold function: Maintaining a critical threshold concentration of BMP at implantation site for the required period (Temporal Distribution) Act as scaffold over which bone growth can occur. Contain the BMP at the localized site and prevent extraneous bone formation. (Spatial containment). Carrier Materials
Natural Polymers: Different collagens, fibrins, Fibronectin, Hyaluronic acids, glycosaminoglycan.
Demineralized Bone matrix.

Synthetic polymers: Poly lactates and poly glycolic acid (PGA) Hyaluronic acid gel

Ceramics: Hydroxyappatite, Tricalcium phosphates. Allograft.

Non ceramic Inorganic material: Calcium phosphate based cements (CPCs), Calcium sulfates, metals and bioglass. Newer delivery models : Depot injectable carriers, viral vectors, gene guns, oral

Similarities / Differences

Scientists isolated the gene for BMP-2 from bone tissue and created genetically engineered cells. These cells then produce large quantities of rhBMP-2. The recombinant form of rhBMP-2 is identical to the natural form in both its chemistry and its ability to grow new bone. The functions are unique for the natural form and recombinant forms Objective BMP-2 rhBMP-2

Chemistry

396 amino acids; 44702 Da

396 amino acids; 44702 Da

Function

ectopic bone formation embryogenesi s regulatory roles voltage-gated K+ channels in human pulmonary artery

Recruits boneforming cells (Mesenchymal stem cells) help heal broken bones.

Structure

Market Facts: Leg Fractures

Fractures of long bones constitute the majority of operating room procedures in most trauma centers (490,000 in U.S. in 2007) Patients with tibial fractures remain in the hospital for a total of 569,000 hospital days and incur 825,000 physician visits per year in the United States

Market Facts: Dental Implants

Approximately 1,000,000 dental implants were performed in 2007.

In addition, the field of oral maxillofacial application (a field that treats a wide spectrum of diseases, injuries and defects in the head, neck, face, jaws and the hard and soft tissues of the oral and maxillofacial region) employ bone fusion technologies as well in a wide variety of surgical techniques

Market Facts: Spinal Fusion

Over 250,000 spinal fusions are performed each year. Average cost of spinal fusion is between $35,000 - $75,000.

Back pain is the most common reason a person takes a day off work, avoid physical activity or exercise. This, in turn, leads to more health problems.
According to the National Institute For Health, Approximately 50% of the population will have evidence of degenerative changes in their cervical spine by the age of 50.

Autogenous Iliac Crest Bone Graft (ICBG)

The Autogenous Iliac Crest Bone Graft surgical technique is the most common method used for bone fusion. There are limitations to this procedure, however. ICBG requires that physicians remove bone from one part of the body and attach it to the fracture of a bone.

But There Are Limitations

There is an increased risk of infection from a second surgical incision. Pain from bone graft site may last from 1-5 days to a year. Additional risks from iatrogenic fracture and damage to nuerovascular structures. Poor bone quality secondary to underlying disease, smoking or medications that effect bone quality. Finally, patient age and limitations to the amount of bone obtained by the illium.

Bone Fusion: The Cost

Bone transplantation procedure can prolong surgery, increase blood loss, increase recovery time. Typical hospital stay for bone fusion is between 4-6 days. 4% of spinal implant patients account for 75% of the cost of caring for back pain.

Introducing INFUSE/InductOs

INFUSE/InductOs (rhBMP-2, dibotermin alfa) Partner: Medtronic (MDT) First license agreement signed in 1995 Complex drug-device combination product Wyeth discovered, developed, and manufactures rhBMP-2 Medtronic is the leader in spinal fusion Product has three U.S. FDA PMA (device) approvals, two EU EMEA MAA (medicinal product) approvals, and additional regional approvals. Further approvals pending. First patent granted for spinal fusion in April 8, 1997. Patent expires April 8, 2014 Leading product in its category Medtronic and Wyeth accomplished alliance and commercial success. Discussions continue on how to foster and expand the relationship. rhBMP-2, which is found naturally in the body in the form of BMP-2, stimulates bone creation rhBMP-2 elimiates the need for bone grafts from hip rhBMP-2 is applied to an absorbable collagen sponge carrier that acts as scaffolding, allowing bone cells to generate in the size and form required to mend bone.

The Benefits of rhBMP-2

Use of rhBMP-2 reduces pain, both short term and long term Reduces number of sick days, days spent in the hospital, physician check-ups. In short, it limits the medical cost of repairing a fractured bone. Few economic benefit analysis studies have been conducted to date, but early studies have been promising: The cost of using rhBMP-2/ACS was $39,967 with a 0.11 mean improvement in SF-6D; and for ICBG the cost was $42,286 with a mean improvement of 0.10 in SF-6D. CONCLUSION: There are more complications, increased need for additional treatment and revision surgery in patients over 60 years old receiving ICBG compared with rhBMP-2/ACS. This may account for higher costs and lower improvements in utility seen in patients INFUSE/InductOs (rhBMP-2, dibotermin alfa) costs $5,000 a unit Over 127,000 units were sold in 2007, with revenues of approximately $635,000,000 Conclusion: Surgeons are willing to accept higher direct costs in exchange for fewer days spent in the hospital, less physical therapy, and faster healing

Bringing A Product To Market

Probability Factors Based on historical numbers, drugs in clinical trials have been approved by the FDA at the following rates: Phase I Trials : 20% Phase II Trials: 30% Phase III Trials: 67% NDA: 85% ( +/- based on different types of drugs)

The percentage of drugs moving from one stage to another: Phase I to Phase II: 67% Phase II to Phase III: 45% Phase III to NDA: 85% NDA to FDA: 85%

Bringing A Product To Market

Cash Flow Difficult to determine. First, costs must be determined. Second, potential revenues must be determined. Costs Phase I (1 year): $500,000 for animal testing plus $12,000 per human subject (20-80 subjects) per

Phase II (1.5 years): $1 million for animals plus $12,000 per human subject ( 100 300)
Phase III (3 years): $1.5 million for animals plus $6,000 per human New Drug Application ( 1 year): $1.8 million Cash flows must be adjusted by probability that they will occur rNPV Cash Flow = ( Revenues x Cost of Revenues x Probability)

Problems with Infuse

According to a recent Cleveland Clinic study, 17% of procedures using Medtronics Infuse bone graft are off label According to the Wall Street Journal, 75% of roughly 300 adverse effects reported to the FDA were off label. Off label use has resulted in numerous lawsuits at Medtronics In November 2007, the U.S. Department of Justice filed a subpoena for Medtronics executives regarding an investigation of kickbacks from the company to surgeons.

Competition

In July 2008, Biocomposites ( a U.K. company) received FDA clearance to market geneX putty.geneX putty is a resorbable bone graft material manufactured through a proprietary process that confers the product with a reproducible negative surface charge. This property stimulates bone cell activity, accelerating bone formation by harnessing key proteins and addressing osteoblast adhesion and proliferation for rapid osteogenesis.