WO2016127253A1 - Method and system for enhancing the activity of bone morphogenetic proteins - Google Patents

Abstract

A method and system for enhancing BMP activity involves the combined administration of a zinc compound with the BMP. The zinc is delivered by a carrier that is adapted to release the zinc, over a period of time, at the site where the BMP is delivered. The concomitant delivery of the zinc compound has been found to enhance the activity of BMP at the delivery site.

Description

METHOD AND SYSTEM FOR ENHANCING THE ACTIVITY

OF BONE MORPHOGENETIC PROTEINS

CROSS REFERENCE TO PRIOR APPLICATIONS

[0001] The present application claims priority under the Paris Convention to US

Application Number 62/113,920, filed on February 9, 2015, the entire contents of which are incorporated herein by reference.

FIELD OF THE DESCRIPTION

[0002] The present description relates to a system and method for enhancing the activity of bone morphogenetic proteins (BMPs). In particularly, the present description relates to a system and method for enhancing the activity of exogenously applied BMP by the addition of an activating compound or agent. More particularly, the description teaches the use of a zinc compound for enhancing BMP activity.

BACKGROUND

[0003] Bone morphogenetic proteins (BMPs) are a family of at least 15 proteins that affect cell migration, proliferation and differentiation. Several BMPs including BMP-2, BMP- 4, BMP-5, BMP-6, and BMP-7 are involved in bone formation, where they promote mesenchymal stem cell (MSC) proliferation and differentiation into osteoblasts and chondroblasts.

[0004] This has led to the evaluation of the exogenous application of these BMPs for the stimulation of bone repair. To be effective, BMPs may be combined with a carrier, which retains the BMP at the implantation site, forming a bioimplant. Examples of the use of carriers to deliver BMPs to a desired location and/or at a desired delivery schedule are provided in US 2014/0100161 and US 2015/0196620, the entire contents of which are incorporated herein by reference.

[0005] Bone morphogenetic protein (BMP) containing bioimplants have been used clinically to promote spine fusion, repair fracture non-unions and bone defects and to augment the bone volume of the alveolar ridge. However these bioimplants require large amounts of BMP to be effective clinically which increases their cost and the risk of adverse events, including an increased immune response and cancer. [0006] Accordingly, there is a need for a bioimplant that requires less BMP to be effective. In other words, there is a need to enhance the activity, or potency of BMPs.

SUMMARY OF THE DESCRIPTION

[0007] In one aspect, the present description provides a system and method for enhancing the activity of BMP which reduces the need to use large amounts of BMP to be effective in promoting tissue repair.

[0008] The present description provides, in one aspect, a system and method for enhancing the activity of bone morphogenetic protein, the system comprising a bone morphogenetic protein, a carrier and a zinc compound wherein the zinc compound is released from the system over a period of time.

[0009] According to another aspect, the carrier comprises particles of calcium sulphate and/or optionally calcium phosphate.

[0010] According to another aspect, the zinc compound may be incorporated into the carrier.

[0011] Thus, in one aspect, there is provided a method of enhancing the activity of at least one bone morphogenetic protein (BMP), the method comprising combining the BMP with a zinc compound.

[0012] In another aspect, there is provided a use of a combination of at least one bone morphogenetic protein (BMP) and a zinc compound in the treatment of a mammal requiring BMP treatment.

[0013] In another aspect, there is provided a system for delivering at least one bone morphogenetic protein (BMP), the system comprising: a first carrier including the at least one BMP; and a second carrier including at least one zinc compound; wherein the first and second carriers are adapted to deliver the at least one BMP and at least one zinc compound, respectively, over a period of time at a treatment site.

BRIEF DESCRIPTION OF THE FIGURES

[0014] The features of certain embodiments will become more apparent in the following detailed description in which reference is made to the appended figures wherein: [0015] Figure 1 shows a histogram indicating the effect of Zinc chloride on rhBMP-2 stimulated alkaline phosphatase activity in C2C12 cells.

[0016] Figure 2 shows a histogram indicating the change in compressive strength of calcium sulphate-zinc beads with increasing zinc chloride weight percent.

[0017] Figure 3 shows scanning electron micrographs of the surface of calcium sulphate-zinc beads with increasing zinc chloride weight percent.

[0018] Figure 4 shows the amount of zinc released into water from calcium sulphate-zinc beads made with different weight percent zinc chloride added.

[0019] Figure 5 shows the total volume of the bone ossicles produced by BMP bioimplants with different amounts of zinc chloride added.

DETAILED DESCRIPTION

[0020] Bone morphogenetic proteins (BMPs) play an integral role in the repair and regeneration of bone and exogenous BMPs can be used to stimulate the repair of bone. For exogenous BMPs to be effective in stimulating repair it is important that they be retained at the site requiring repair and be protected from inactivation, sequestration or degradation. As discussed above, to achieve this, it has been proposed that the BMP be combined with a carrier, wherein the carrier and BMP combination is implanted at a desired treatment site. In some cases, the amount of BMP required to be effective is very high thereby greatly increasing the cost and risk of adverse events associated with a high BMP concentration.

[0021] Zinc is an essential trace metal and has been shown to stimulate bone growth and osteoblast proliferation and has been incorporated into biomaterials to stimulate bone healing (Y. Qiao et al., Stimulation of Bone Growth Following Zinc Incorporation Into Biomaterials, Biomaterials, 35 (25), 2014, p.6882-6897). However, no attempt has been made to enhance BMP activity by the addition or combined use of zinc.

[0022] The present description is based on the present inventors' findings that, inter alia, the inclusion of a zinc compound with a carrier of BMPs improves the activity of such BMPs and therefore the BMP-containing carrier, or bioimplant.

[0023] The system and method of the invention can be used for a variety of therapeutic and clinical applications, including: fracture repair; bone grafts; spine fusion; and regeneration of skull, mandibular, and bone defects. For such applications, the system of the invention is preferably provided on, or in the form of a bioimplant.

[0024] Definitions

[0025] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs

[0026] As used herein the term "bioimplant" refers to a material which is suitable for implantation and contains an exogenous growth, or biologically active factor.

[0027] The term "recombinant protein" is meant to refer to a protein produced by a transiently transfected, stably transfected, or transgenic host cell or animal as directed by an expression construct containing the cDNA for that protein. This term also encompasses pharmaceutically acceptable salts of such a protein.

[0028] As used herein, the term "polypeptide" or "protein" refers to a polymer of amino acid monomers that are alpha amino acids joined together through amide bonds.

Polypeptides are therefore at least two amino acid residues in length, and are usually longer. Generally, the term "peptide" refers to a polypeptide that is only a few amino acid residues in length. A polypeptide, in contrast with a peptide, may comprise any number of amino acid residues. Hence, the term polypeptide included peptides as well as longer sequences of amino acids.

[0029] The terms "bone morphogenetic protein" or "bone morphogenic protein" or "BMP" may be used interchangeably and refer to any member of the bone morphogenetic protein (BMP) subfamily of the transforming growth factor beta (TGFp) superfamily of growth and differentiation factors, including BMP-2, BMP-3 (also known as osteogenin), BMP-3b (also known as growth and differentiation factor 10, GDF-10), BMP-4, BMP-5, BMP-6, BMP-7 (also known as osteogenic protein-1 , OP-1), BMP-8 (also known as osteogenic protein-2, OP-2), BMP-9, BMP-10, BMP-1 1 (also known as growth and differentiation factor 8, GDF-8, or myostatin), BMP-12 (also known as growth and differentiation factor 7, GDF-7), BMP-13 (also known as growth and differentiation factor 6, GDF-6), BMP-14 (also known as growth and differentiation factor 5, GDF-5), and BMP-15.

[0030] The term "bone morphogenetic protein", or "BMP", also encompasses allelic variants of BMPs, function conservative variants of BMPs, and mutant BMPs that retain BMP activity. The BMP activity of such variants and mutants may be confirmed by any of the methods well known in the art including stimulation of alkaline phosphatase activity in muscle derived C2C12 cells (Peel et al. J Craniofac Surg. 2003 May; 14(3):284-91) or marrow derived W20-17 cells (Thies et al. Endocrinology. 1992 Mar; 130(3): 1318-24) or induction of ectopic bone formation when implanted in muscle in vivo (Barr et al. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2010 Apr; 109(4):531 -40).

[0031] In one aspect, the BMP used in the system and method described herein may be one or more of BMP-2, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8 and BMP-9. In one aspect, the BMP is one or more of BMP-2, BMP-4 and BMP-7.

[0032] In one aspect the BMP is a mammalian BMP (e.g., mammalian BMP-2 or mammalian BMP-7). In one aspect, the BMP is a human BMP (hBMP) (e.g. hBMP-2 or hBMP-7).

[0033] As used herein the term "carrier" refers to a material comprising single or multiple components and is adapted to retain and deliver BMPs and/or zinc compounds at a treatment site over a period of time. In one aspect, the period of time over which the BMPs and/or zinc compounds are retained and delivered at the treatment site is between several days and several weeks. Preferably, the carrier is adapted to retain and deliver the BMPs and/or zinc compounds for a period of weeks.

[0034] The terms "comprise", "comprises", "comprised" or "comprising" may be used in the present description. As used herein (including the specification and/or the claims), these terms are to be interpreted as specifying the presence of the stated features, integers, steps or components, but not as precluding the presence of one or more other feature, integer, step, component or a group thereof as would be apparent to persons having ordinary skill in the relevant art.

[0035] The present inventors have shown that zinc compounds can be combined with carriers that are known for delivering BMPs at a desired treatment site within a body of a subject. The inventors have also shown that such carriers can deliver zinc at the treatment site. Finally, the inventors have shown that the presence of zinc enhances BMP activity. Based on the findings outlined herein, it is believed that the presently described system and method can be used for any BMP or combination of BMPs and with a wide variety of zinc compounds. [0036] Various BMPs have been discussed above and many have been used for clinical administration. Aspects of the present description have been illustrated with the use of ZnCI (as indicated in the following examples). However, as illustrated in Example 1 , Zn⁺⁺ ions are found to have the required BMP activity enhancing feature. It will therefore be understood that while the description has been illustrated with one particular zinc compound, a variety of other zinc compounds may also be used in the present system and method for enhancing BMP activity. In this regard, any zinc compound that is suitable for implantation in an animal's body can be used. Another criterion in choosing the zinc compound is the ability of the compound to be released when combined with carrier. Without limiting the scope of the description in any way, the following are examples of Zn compounds that may be used in the system and method taught herein: zinc chloride, zinc acetate, zinc sulphate, zinc acexamate, and zinc DL-aspartate.

[0037] As described herein, the zinc compounds used for enhancing BMP activity are preferably combined with a carrier, which may be the same carrier onto which the BMPs are provided. However, it will be understood that the zinc compounds may also be provided on one or more different carriers. In this regard, such zinc compound carrier(s) may include: zinc-releasing calcium phosphate ceramics (as described by A. Ito et al., Materials Science and Engineering, C 22 (2002) 21-25); zinc releasing glasses (as described by Adel et al. , J. Mat. Sci: Mater Med (2015) 26: 151 ); or zinc releasing calcium phosphate cements. The present description is not limited to any particular carrier.

[0038] In one aspect, the zinc compound is provided on the carrier or carrier system at a concentration of less than or equal to about 1 wt%. Preferably, the zinc compound is provided at a concentration of between about 0.1 and about 1 wt%. In one aspect, the zinc compound is provided at a concentration of about 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1 wt%. In one aspect, the carrier or carrier system is designed to release zinc over a period of time (i.e. in a delayed release profile). More generally, the carrier is designed to release zinc at a concentration that is sufficient to enhance BMP activity while not being at toxic levels to the animal being treated.

[0039] The carriers for BMPs and/or zinc compounds may be any known carrier that is used for delivering compounds (such as medicaments etc.) to a delivery site in the body of a mammal. In one aspect, the carriers comprise particles. In one aspect, the particles are calcium sulphate particles or a combination of calcium sulphate and calcium phosphate particles. [0040] Kits

[0041] In one aspect, there is provided a kit for containing the system described herein. The kit preferably comprises a container comprising the carrier onto which may be loaded or coated the associated BMP or BMPs and zinc compound(s).

[0042] In one aspect, the kit described herein may comprise any necessary reagents and/or instruments and/or instructions and/or vessels as may be needed.

[0043] Examples

[0044] The invention is next described by means of the following examples. It will be understood that the following examples are intended to be illustrative only, and do not in any way limit the scope of the present description.

[0045] Example 1 : Screening Additives To Determine Which Enhance BMP Activity

[0046] The ability of various additives to enhance BMP activity was evaluated by testing their ability to enhance BMP stimulated alkaline phosphatase (ALP) activity in the C2C12 assay described by Peel et al. J Craniofacial Surg. 2003; 14:284-291 .

[0047] C2C12 cells were seeded into 24 well plates and allowed to attach for at least 2 hours, after which the media was removed. Fresh media with or without rhBMP-2 and the test additives, zinc chloride (0 to 200 μΜ), strontium chloride (0 to 2mM), calcium chloride (0 to 1 mM) or sodium silicate (0 to 100 μΜ) was added to the cells. After 48 to 72 hours the cells were lysed and the ALP activity was measured using a colorimetric assay.

[0048] The presence of any of the additives at the concentrations tested did not stimulate ALP activity in the absence of rhBMP-2. The addition of rhBMP-2 stimulated ALP activity in C2C12 cells. The further addition of strontium chloride, calcium chloride or sodium silicate did not further increase the rhBMP-2 stimulated ALP activity. When zinc chloride was added there was a significant further increase in the rhBMP-2 stimulated ALP activity (Figure 1).

[0049] It is noted that this example involved the testing of Zn, Sr, Si and Ca compounds. While all of such compounds are known to stimulate bone growth, this study shows that only the Zn compound was found to enhance BMP activity. [0050] Example 2: Preparing A Calcium Sulphate Carrier Containing A Zinc Compound

[0051] The results from Example 1 indicated that addition of zinc chloride to the culture media enhanced the activity of BMP while the other test compounds did not. We therefore wished to prepare a BMP carrier that could incorporate zinc chloride and release it over time once placed in solution. For the first part of this study we determined whether we could incorporate zinc chloride into calcium sulphate dihydrate beads and what the effect of varying the concentration of zinc chloride would be on the beads.

[0052] Zinc chloride (anhydrous powder) was mixed with calcium sulphate hemihydrate (Ca₂H₂0₉S₂), referred to herein as "CaSH", and deionized water according to the ratios in Table 1.

[0053] Table 1

[0054] Samples were mixed in a mixing bowl using a spatula for 30 to 40 seconds and the resultant paste was placed into molds (Wright Bead Template; Wright Medical). After 2 minutes the setting of the calcium sulphate zinc (referred to herein as "CaS-Zn") beads was evaluated manually by determining if it was possible to produce an indentation with a gentle application of the tip of a probe. The beads were tested every minute until the sample could no longer be indented and the setting time was recorded. The samples were left in the mold for a further 24 hours before being removed. They were then stored at room temperature until use. [0055] The compressive strength of the beads was determined using an Instron™ testing system. The setting time and compressive strength results were evaluated for differences statistically using Sigma Plot™ software. The topography of the beads was determined by scanning electron microscopy.

[0056] Results indicated that with increasing zinc concentration the setting time (not shown) and compressive strength (Figure 2) was reduced while the crystal size appeared to increase (Figure 3).

[0057] Example 3: Testing The Carrier Containing Zinc For Release Of Zinc

[0058] The results from Example 2 indicate that we are able to incorporate zinc chloride into calcium sulphate dihydrate beads. This example was conducted to demonstrate that zinc could be released from the beads over time once placed in the body.

[0059] CaS-Zn pellets were prepared with weight percent zinc chloride added of up to 5% as described in Example 2. Beads were weighed individually and placed in separate 2ml_ centrifuge tubes. 1 ml_ of culture media (alpha minimal essential medium + 10% fetal bovine serum) was added to each tube, which was then placed on a rotary shaker at 38°C for 7 days. The media was removed and placed in clean centrifuge tubes, centrifuged and the supernatant collected. The zinc released from the CaS-Zn pellets was estimated by measuring the zinc concentration in the supernatants using a colorimetric zinc assay (Sigma Aldrich) according to the manufacturer's instructions.

[0060] The results of this testing indicated that measureable amounts of zinc were released over 7 days from the scaffolds with a weight percent of zinc chloride of 0.05% and above as shown in Figure 4.

[0061] Example 4: Testing BMP Bioimplants Containing Zinc For Activity In Vivo

[0062] This example was conducted to determine whether the incorporation of zinc chloride into a BMP carrier that releases zinc would increase the amount of bone formed.

[0063] Calcium sulphate beads were prepared with either 0, 0.1 or 1 % of ZnCI₂ added as described in example 2. The beads were placed in #5 gelatin capsules and sterilized by a 25kGy dose of gamma irradiation.

[0064] The gelatin capsules containing the beads were then implanted into a biceps femoris of a mouse as described by Barr et al. (Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontics. 2010; 109:531-540) and 30μg of recombinant human BMP-2 (rhBMP-2) was pipetted onto the beads. On the contralateral side gelatin capsules with CAS-Zn beads were implanted but no rhBMP-2 was added to the beads. The overlying skin was closed with mitchel clips.

[0065] After 28 days the mice were sacrificed and the hind limbs were fixed in 10% formalin solution.

[0066] After fixation the volume of bone that was formed in the muscle pouch was evaluated by microcomputed tomography. Following this the samples were decalcified and processed for histological evaluation.

[0067] The results indicated that the newly induced bone ossicles formed by the BMP bioimplants that included zinc had a greater total volume than those of the control bioimplants without any zinc added (Figure 5). Histological evaluation confirmed that the masses formed were bony ossciles and that no sign of cytotoxicity was observed.

[0068] Although the above description includes reference to certain specific

embodiments, various modifications thereof will be apparent to those skilled in the art. Any examples provided herein are included solely for the purpose of illustration and are not intended to be limiting in any way. Any drawings provided herein are solely for the purpose of illustrating various aspects of the description and are not intended to be drawn to scale or to be limiting in any way. The scope of the claims appended hereto should not be limited by the preferred embodiments set forth in the above description, but should be given the broadest interpretation consistent with the present specification as a whole. The disclosures of all prior art recited herein are incorporated herein by reference in their entirety.

Claims

WE CLAIM:

1 . A method of enhancing the activity of at least one bone morphogenetic protein (BMP), the method comprising combining the BMP with a zinc compound.

2. The method of claim 1 , wherein the BMP and zinc compound are delivered together at a treatment site.

3. The method of claim 1 or 2 wherein the BMP and the zinc compound are provided on the same or different carriers and wherein the carriers are adapted to deliver the BMP and the zinc compound.

4. The method of claim 3, wherein the carriers release the BMP and the zinc compound over a period of time.

5. The method of claim 4, wherein the period of time is days or weeks.

6. The method of any one of claims 3 to 5, wherein the carrier comprises particles of calcium sulphate and/or calcium phosphate.

7. The method of any one of claims 3 to 6, wherein the zinc compound is provided on the carrier at a concentration of about 0.1 to about 1 wt%.

8. The method of any one of claims 1 to 7, wherein the BMP is BMP-2, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9, or a combination thereof.

9. The method of any one of claims 1 to 8, wherein the zinc compound is zinc chloride, zinc acetate, zinc sulphate, zinc acexamate, zinc DL-aspartate, or a combination thereof.

10. The method of any one of claims 1 to 9, wherein the at least one BMP and the zinc compound are administered to a mammal requiring BMP treatment.

1 1 . A use of a combination of at least one bone morphogenetic protein (BMP) and a zinc compound in the treatment of a mammal requiring BMP treatment.

12. The use of claim 1 1 , wherein the BMP and zinc compound are on the same or different carriers and wherein the carriers are adapted to deliver the BMP and the zinc compound.

13. The use of claim 12, wherein the carriers are adapted to release the BMP and the zinc compound over a period of time.

14. The use of claim 13, wherein the period of time is days or weeks.

15. The use of any one of claims 1 1 to 13, wherein the carrier comprises particles of calcium sulphate and/or calcium phosphate.

16. The use of any one of claims 1 1 to 15, wherein the zinc compound is provided on the carrier at a concentration of about 0.1 to about 1 wt%.

17. The use of any one of claims 1 1 to 16, wherein the BMP is BMP-2, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9, or a combination thereof.

18. The use of any one of claims 1 1 to 17, wherein the zinc compound is zinc chloride, zinc acetate, zinc sulphate, zinc acexamate, zinc DL-aspartate, or a combination thereof.

19. A system for delivering at least one bone morphogenetic protein (BMP), the system comprising: a first carrier including the at least one BMP; and a second carrier including at least one zinc compound; wherein the first and second carriers are adapted to deliver the at least one BMP and at least one zinc compound, respectively, over a period of time at a treatment site.

20. The system of claim 19, wherein the first and second carriers are the same.

21. The system of claim 19 or 20, wherein the period of time is days or weeks.

22. The system of any one of claims 19 to 21 , wherein the first and second carriers comprise particles of calcium sulphate and/or calcium phosphate.

23. The system of any one of claims 19 to 22, wherein the zinc compound is provided on the second carrier at a concentration of about 0.1 to about 1 wt%.

24. The system of any one of claims 19 to 23, wherein the BMP is BMP-2, BMP-4, BMP- 5, BMP-6, BMP-7, BMP-8, BMP-9, or a combination thereof.

25. The system of any one of claims 19 to 24, wherein the zinc compound is zinc chloride, zinc acetate, zinc sulphate, zinc acexamate, zinc DL-aspartate, or a combination thereof.

26. The system of any one of claims 19 to 25, wherein the system comprises a bioimplant.

27. A use of the system of any one of claims 19 to 26 in the treatment of a mammal in need of BMP treatment.