WO2016025496A1

WO2016025496A1 - Platelet factor 4 for use in the treatment of bone diseases

Info

Publication number: WO2016025496A1
Application number: PCT/US2015/044681
Authority: WO
Inventors: Zsombor Lacza; István HORNYÁK
Original assignee: Lacerta Technologies Inc.
Priority date: 2014-08-11
Filing date: 2015-08-11
Publication date: 2016-02-18

Abstract

The invention relates to novel uses of Platelet Factor 4 (PF-4), methods for treatment bone diseases and compositions comprising PF-4. The invention also relates to uses of PF-4 in the preparation of implants.

Description

PLATELET FACTOR 4 FOR USE IN THE TREATM ENT OF BONE DISEASES

FIELD OF THE INVENTION

BACKGROUND ART

Platelet factor 4 (PF-4 or PF4) is a small cytokine belonging to the CXC chemokine family that is also known as chemokine (C-X-C motif) ligand 4 (CXCL4). PF-4 promotes blood coagulation by moderating the effects of heparin-like molecules and forms a complex with proteoglycan and shares homologies in particular with β-thromboglobulin and interleukin-8 (IL-8) [Jouan, V et al., "Inhibition of In Vitro Angiogenesis by Platelet Factor-4-Derived Peptides and Mechanism of Action" Blood, Vol 94, No 3, 1999]. This chemokine in mammals is released from alpha-granules of activated platelets during platelet aggregation.

The precursor of PF-4 is, together with the signal peptide, a 101 amino acid precursor protein in humans. Once the signal peptide has been cleaved a 70 amino acid peptide (amino acids 32-101, numbering based on the pre-peptide) is formed. In this application, unless noted otherwise, the numbering of this normal or long form is used (1-70). There is a short form of the peptide having amino acids 17-70 (or 48-101 based on the pre-peptide numbering) [see uniprot entry UniProtKB - P02776 (PLF4_HUMAN)].

Its major physiologic role appears to be neutralization of heparin-like molecules on the endothelial surface of blood vessels, thereby inhibiting local antithrombin III activity and promoting coagulation. As a strong chemoattractant for neutrophils and fibroblasts, PF4 probably has a role in inflammation and wound repair [Eisman R, Surrey S, Ramachandran B, Schwartz E, Poncz M (July 1990). "Structural and functional comparison of the genes for human platelet factor 4 and PF4alt". Blood 76 (2) : 336- 44.]

PF-4 is probably localized in other stromal components such as lymphocytes and endothelial cells, and interacts with a variety of immune cells like monocytes and neutrophils. Therefore, PF-4 is expected to exert important pathophysiological effects against solid tumors and infections. Furuya M. et al. have found that PF4 and its receptor CXCR3B were downregulated in clear cell ovarian cancers and in tumor-associated macrophages (TAMs) of ovarian cancers arising in endometriosis [Furuya M, et al. "Differential expression patterns of CXCR3 variants and corresponding CXC chemokines in clear cell ovarian cancers and endometriosis" Gynecol Oncol 122, 2011 648-55. and Furuya M et al. "Impaired CXCL4 expression in tumor-associated macrophages (TAMs) of ovarian cancers arising in endometriosis" Cancer Biology & Therapy 13:8, 671-680] .

By Lampert M P et al. it has been found that PF-4 or a high level thereof may increase sensitivity to develop thrombocytopenia after bone marrow injury and inhibiting PF4 may be beneficial in this case [Lampert M P et al. "Platelet factor 4 is a negative autocrine in vivo regulator of megakaryopoiesis: clinical and therapeutic implications" Blood. 2007; 110:1153-1160).

Prior art considerations on the potential clinical uses of PF-4 are summarized by Lippi G and Favoloro EJ [Lippi G and Favoloro EJ "Recombinant platelet factor 4: a therapeutic, anti-neoplastic chimera?" Semin Thromb Hemost. 36(5) 2010 558-69].

A variety of growth factors have been found to play a role in bone healing. The two possibly most important families can be classified as the Bone-Derived Growth Factors, namely the BMPs family, which are mainly used for bone regeneration, and the Autologous Blood-Derived Growth Factors, which are thought to be effective in cartilage regeneration [Civinini R. et al. "Growth factors in the treatment of early osteoarthritis" Clinical Cases in Mineral and Bone Metabolism 26 2013; 10(1) : 26- 29].

In particular, BMP2 to BMP7 belong to the Transforming growth factor beta superfamily of proteins; among them, BMP2 and BM P7 appear to be particularly effective in bone formation and osteoblast differentiation. For example, recombinant human BMP2 (rhBMP-2, contained in InFuse Bone Graft) has received premarket approval for fusion of the lumbar spine in skeletally mature patients with degenerative disc disease (DDD) with some restrictions, and rhBMP-7 (referred to as OP-1) has received humanitarian device exemption approval as an alternative to autograft in recalcitrant long bone nonunions where use of autograft is unfeasible and alternative treatments have failed and in some other conditions. However, the FDA warns that the safety and effectiveness of rhBM P in the cervical spine have not been demonstrated and life-threatening adverse effects like swelling of neck and throat tissue with severe consequences may occur [Schultz, Daniel G. "Life-threatening Complications Associated with Recombinant Human Bone Morphogenetic Protein in Cervical Spine Fusion" FDA Public Health Notifications (Medical Devices), July 1, 2008, http://www.fda.gov/MedicalDevices/Safety/AlertsandNotices/PublicHealthNotifications/ucm062000.htm ] . A review on the medical use of Bone morphogenetic proteins has been authored by Bessa PC et al. [Bessa PC et al. "Bone morphogenetic proteins in tissue engineering: the road from the laboratory to the clinic, part I (basic concepts)" J Tissue Eng Regen Med. 2(1) 2008 1-13] .

It has also been suggested that certain growth factors present in the platelets, like PDGF and others may have a specific activity on neoproliferation, specifically on cartilage and bone regeneration [Civinini R. et al., supra].

PDGF (Platelet-derived Growth Factor) apparently initiates connective tissue healing through the promotion of collagen and protein synthesis, and has a mitogenic activity to mesoderm-derived fibroblasts and chondrocytes. Assumed use of PDGF in cartilage repair is based on its role in wound healing and stimulation of matrix synthesis in growth plate chondrocytes [Fortier LA et al., "The role of growth factors in cartilage repair" Clin Orthop Relat Res. 469(10) 2011 2706-15] .

Many growth factors derived from blood are released upon platelet activation, and therefore their clinical use in the form of Platelet-rich plasma (PRP) has been proposed. [Civinini R. et al ., supra]. Ischemic and arthritic condition of hardly regenerating tissues, in particular the cartilage or the bone is a serious problem affecting an increasingly larger population. For example, arthritis of the bone typically develops over decades, offering a long window of time to potentially alter its course. Early diagnosis of an increased risk of development this disease may facilitate early treatment as well and to prevent late stage clinical manifestation of the disease.

Ischemic condition of the bone and cartilage may occur upon injuries or during surgery, wherein improvement in the regeneration of the cartilage or the bone is required.

Thus, there is still a need in the art to regenerate bone and cartilage tissue and to find ways of treatment of these and similar conditions.

BRIEF DESCRIPTION OF THE INVENTION

In a first aspect the invention relates to a method of treating a patient in need of treatment with any of BMP-2, BMP-7, PDGF or PF-4 due to the condition of said patient, by administering an effective amount of a serum fraction of platelet rich fibrin (SPRF). The invention also relates to a method of treating a patient in need of treatment with PF-4 due to the condition of said patient by administering an effective amount of a serum fraction of platelet rich fibrin.

In a further aspect the invention relates to a method of treating a patient being in need of bone regeneration or bone formation due to a condition of said patient, said method comprising administration of an effective amount of PF-4 to said patient.

In an embodiment the invention relates to a method of treating a patient having a condition wherein bone cells of said patient have suffered or been exposed to a low leveloxygen and/or a low level of- glucose (or a condition modeled by oxygen-glucose deprivation (OGD)) said method comprising administration of an effective amount of PF-4 to said patient.

In an embodiment the invention relates to a method of treating a patient having a condition wherein bone cells of said patient are or have been exposed to an ischemia, said method comprising administration of an effective amount of PF-4 to said patient.

In a preferred embodiment of the invention the condition is selected from degenerative bone diseases. In a preferred embodiment of the invention the condition is selected from bone ischemia or a related bone disease, bone necrosis (osteonecrosis), osteoarthrosis and osteoarthritis or other related bone disease. In a preferred embodiment of the invention the condition is selected from ischemic bone damage, ischemic bone disease, ischemic bone necrosis, avascular necrosis, metabolic bone disease, or any bone disease in which death or insufficient propagation of bone cells (osteocytes) occurs. Preferably, the patient is in need of facilitating or accelerating the propagation of bone tissue cells and thereby bone tissue regeneration. In an embodiment the condition is selected from bone injury, bone graft and bone fracture. For example the patient has been the subject of a bone graft.

More preferably, said need of the patient is due to bone ischemia or a disease which is a consequence of bone ischemia or a disease mediated by bone ischemia. Accordingly, the invention further relates to a method for the treatment of a patient suffering in or having suffered in osteoarthritis, osteoarthrosis, bone necrosis, bone ischemia, or a disease as defined herein or hereinabove, comprising the steps of administering a PF-4 to said patient. The invention also relates to a method for the treatment of a patient suffering in or having suffered in bone injury, bone graft or bone fracture.

In a further aspect the invention relates to a method or use of PF-4 for facilitating or promoting the propagation of bone tissue cells and thereby bone tissue regeneration comprising the step of administering PF-4 to a bone tissue under or having subjected to bone ischemia.

Preferred methods according to the invention - either in vitro or in vivo - refer to restoring the proliferation capacity of post-ischemic bone. In a further preferred variant bone regeneration or bone formation is accompanied by simultaneously promoting wound healing by PF-4.

In the method of the invention an effective amount of PF-4 is typically an amount sufficient to treat, repair or augment cells or tissue, e.g. local or topical treatment at a target site in need of cell proliferation or regeneration of impaired bone, e.g. at a site of a wound, an injury, an incision, or a surgical site.

In an embodiment PF-4 is administered in a dosage resulting in supraphysiological concentration of PF-4 in the body of the patient.

In an embodiment PF-4 is administered in a dosage resulting in supraphysiological concentration at a target site in need of treatment, in particular in the local environment of the bone to be treated. In a further embodiment of the invention PF-4 is present in composition obtained from its natural environment wherein several growth factors are present in said composition. In an embodiment no supraphysiological doses are needed.

In a preferred embodiment the PF-4 is recombinant PF-4.

In a preferred embodiment the patient is a human and PF-4 is recombinant human PF-4.

In an alternative embodiment PF-4 is present in a serum fraction. In a preferred embodiment PF-4 is present in a serum fraction selected from a plasma fraction preparation or composition, e.g. platelet poor plasma or serum fraction of platelet rich fibrin (SP F) preparation or composition.

In an embodiment of the invention, there is further provided a method or use of promoting in vitro proliferation of cells by contacting said cells with PF-4 and incubating said cells for a period of time sufficient to promote cell growth or regeneration, wherein the cells are selected from bone cells, in particular osteocytes, osteoclasts, osteoblasts; and bone marrow derived cells such as mesenchymal stem cells and progenitor cells derived from them. In a particular embodiment autogenous bone material or allografts are prepared.

In particular the invention relates to an in vitro method for using PF-4 or use of PF-4 as a cell culture additive or to prepare bones or implants, said implants comprising PF-4. For example said implants can be usual bone implants having PF-4 on their surface applied by surface treatment or coating. For example, PF-4 is used in preparing bone grafts support, e.g. dental bone grafts support, hip joint, etc. In a still further aspect of the present invention the invention relates to PF-4 for use in the treatment of a patient in need of treatment with any of BMP-2, BMP-7, PDGF or PF-4 due to the condition of said patient. The invention also relates to PF-4 for use in the treatment of a patient in need of treatment with PF-4 due to the condition of said patient.

In a further aspect the invention relates to PF-4 for use in the treatment of a patient in need of bone regeneration or bone formation due to the condition of said patient.

The invention also relates to PF-4 for use in the treatment of a patient having a condition wherein bone cells of said patient have suffered or been exposed to a low leveloxygen and/or a low level of- glucose (or a condition modeled by oxygen-glucose deprivation (OGD)).

The invention also relates to PF-4 for use in the treatment of a patient having a condition wherein bone cells of said patient are of have been exposed to ischemia.

In a preferred embodiment of the invention the condition of said patient in which PF-4 is for use in the treatment of is selected from degenerative bone diseases. In a preferred embodiment of the invention the condition is selected from bone ischemia or a related bone disease, bone necrosis, osteoarthrosis and osteoarthritis or other related bone disease. In a preferred embodiment of the invention the condition is selected from ischemic bone damage, ischemic bone disease, ischemic bone necrosis, avascular necrosis, metabolic bone disease, or any bone disease in which death or insufficient propagation of bone cells (osteocytes) occurs. Preferably, the patient is in need of facilitating or accelerating the propagation of bone tissue cells and thereby bone tissue regeneration. In a further aspect the invention PF-4 is for use in facilitating or promoting the propagation of bone tissue cells and thereby bone tissue regeneration in a patient having a condition as defined herein. Preferably PF-4 is for or is used for restoring the proliferation capacity of post-ischemic bone and simultaneously promoting wound healing.

In the method of the invention an effective amount of PF-4 is as defined above in the definition of the methods of the invention or in the appended claims.

In a further embodiment of the invention PF-4 is present in composition obtained from its natural environment wherein several growth factors are present in said composition. In an embodiment no supraphysiological doses are needed.

In a preferred embodiment the PF-4 is recombinant PF-4.

In an alternative embodiment PF-4 is present in a serum fraction. In a preferred embodiment PF-4 is present in a serum fraction composition selected from e.g. platelet poor plasma or serum fraction of platelet rich fibrin (SP F) preparation or composition. BRIEF DESCRIPTION OF THE FIGURE

Figure 1 shows the effect of PF-4 in comparison with growth factors and SPRF on bone explants after OGD. Human bone explants were subjected to oxygen-glucose deprivation (OGD) for 7 hours and then further cultured under normal cell culture conditions for 6 days. Cell viabilty is measured by MTT assay. The OGD treated cells were unable to proliferate and remained at a very low level ('OGD'), whereas treatment with PF-4 and Serum from Platelet Rich Fibrin ('SPRF') restored proliferation capacity, although not to the same level as the controls that did not undergo OGD ('control'). Addition of recombinant PF-4 had a similar effect than that of SPRF or recombinant growth factors. Interestingly, the effect of PDGF was dose dependent in an unusual fashion, i.e. the lower and the higher doses were more effective than the medium dose.

Explants were treated during OGD and continuously for 6 days after OGD with either SPRF or recombinant growth factors in various concentrations. On the 6th day the medium was changed and cell viability assay was done.

DETAILED DESCRIPTION OF THE INVENTION

Platelet Factor 4

This gene encodes a member of the CXC chemokine family. This chemokine is released from the alpha granules of activated platelets in the form of a homotetramer which has high affinity for heparin and is involved in platelet aggregation. This extracellular protein is chemotactic for numerous other cell type and also functions as an inhibitor of hematopoiesis, angiogenesis and T-cell function.

The amino acid sequence of human Platelet Factor 4 (PF-4) has been described by Deuel, Thomas F in 1977 [Deuel, TF et al. "Amino acid sequence of human platelet factor 4" Proc. Natl. Acad. Sci. USA Biochemistry, 74(6), 2256-2258, 1977]. Since then PF-4 from a number of other sources have been indentified and sequenced, available via Gene database of the National Center for Biotechnology Information, U.S. National Library of Medicine 8600 Rockville Pike, Bethesda MD, 20894 USA, GenelD: 5196 (http://www.ncbi.nim.nih.gov/gene/7Term-ortholog gene 5196fgroup1).

Sequence and data of human PF-4 (PLF4_HUMAN) can be found at entry P02776 of the UniProt Knowledgebase (UniProtKB, http://www.uniprot.org/help/uniprotkb) [Jain E. et al. "Infrastructure for the life sciences: design and implementation of the UniProt website" BMC Bioinformatics, 10:136 (2009)].

Features of the protein are disclosed in the UniProtKB database.

Also sequence variants and fragments as well as those of orthologs are described here. Orthologs can be searched e.g. by the BLAST algorithm and service of the database. Such variants or fragments can be found in each entries.

The skilled person will understand as evident that all orthologs, sequence variants and fragments active in a bone cell viability test as disclosed herein, in particular any fragment comprising at least the short form of the peptide (typically having amino acids 17-70 or 48-101 based on the pre-peptide sequence or in particular any fragment occurring in nature, are categorized as PF-4 and can be used in the present invention provided that they are functional, in particular as far as they restore viability of bone cells after oxygen glucose deprivation (OGD), e.g. in a cell viability assay (e.g. methyl-thiazol- tetrasolium (MTT) assay) as described in Example 2. Platelet Factor 4 can be obtained from among others Haematologic Technologies, Inc. (57 River Road, Unit 1021 Essex Junction, Vermont 05452 USA).

It has been known that Platelet Factor 4 is present in the serum [Poruk, E. Katherine et al. "Serum Platelet Factor 4 Is an Independent Predictor of Survival and Venous Thromboembolism in Patients with Pancreatic Adenocarcinoma" Cancer, Epidemiology, Biomarkers & Prevention 19(10) 2010, 2605-2610.]. It occurs in various serum fractions at different level (see Table 1 below).

Preparation of recombinant PF-4

PF-4 was cloned and expressed at first by Eisman et al. in 1990 [Eisman et al., supra]. As the coding sequence of the human and several non-human mammalian PF-4 gene is known it is well within the skills of a person skilled in the art to clone, express and isolate any recombinant PF-4.

Recombinant high level bacterial production of functional human PF-4 is taught by Myers JA et al. [Myers JA et al. "Expression and purification of active recombinant platelet factor 4 from a cleavable fusion protein" Protein Expression and Purification 2(2-3) 1991, 136-143] who describe that alarge- scale production of PF-4 in E. coli, one that might be applied to large-scale production of PF4 protein for possible clinical application. Functionality of the so produced and purified His-PF-4 protein was further identified by cleavage with enterokinase and MS, and its heparin-neutralizing activity was determined by colony formation assay [The skilled person will recognized that other well known expression systems, e.g. bacterial or mammalian expression systems exist in which this small protein can produced in a fully functional manner.

Preparation and means of delivery of recombinant platelet factor 4 is summarized by Lippi G and Favoloro EJ [Lippi G and Favoloro EJ "Recombinant platelet factor 4: a therapeutic, anti-neoplastic chimera? Semin Thromb Hemost. 36(5) 2010 558-69].

PF-4 assays

The level of PF-4 can be measured by ELISA, e.g. as described in Poruk et al (Poruk et al., supra) wherein serologic PF4 measurements were done by ELISA ("ELISA for platelet factor 4", Asserchrom, Stago, Mount Olive, NJ, USA).

A Heparin Platelet Factor 4 (HIT) Assay is also available from University of Florida, Health Pathology Laboratories, USA (http://pathlabs.ufl.edu/tests/heparin-plateiet-factor-4-assay).

The skilled person will understand, however, that any method for assaying the PF-4 level is applicable in the present invention.

Animal Model

In Pf4-null mice which does not express PF-4 and transgenic mice that overexpressed human PF4, Lambert et al. (2007) [Lambert, M. P., Rauova, L, Bailey, M., Sola-Visner, M. C, Kowalska, M. A., Poncz, M. Platelet factor 4 is a negative autocrine in vivo regulator of megakaryopoiesis: clinical and therapeutic implications. Blood 110: 1153-1160, 2007] have been created and disclosed. These animal models can be used by a person skilled in the art to set circumstances and regime of an actual treatment.

Treatment options, preparations and formulations The different possible formulations according to the present invention may be prepared for therapeutic purposes, as needed, for permitting the administration of therapeutic effective amounts of PF-4 thereof to a patient in need thereof.

The formulation according to the present invention may be used to treat human or mammalian patients. The term "patient" includes human and other mammalian subjects that receive therapeutic treatment. The term "treatment" is thus meant to include both prophylactic and therapeutic treatment, in particular to treat, repair or augment a tissue at a target site.

The term "administration" as used herein shall include routes of introducing or applying activated a preparation, such as PF-4, optionally present in a composition e.g. a serum fraction of the invention, to a subject in need thereof to perform their intended function.

PF-4 can be administered systemically e.g. in the form of an injection, tubing etc. In this setting, intravenous administration is preferred. Various known delivery systems, including syringes, needles, tubing, bags, etc., can be used.

Alternatively, preferred routes of administration are local, including application to a wound site or a site of (surgical) intervention, such as by using a fluid, spray, hydrogel or else by any other convenient route, e.g. sprayed onto tissue surfaces, mixed with bodily fluids, etc. Specific delivery systems employ patches for topical delivery, or implants.

PF-4 can be administered, as far as possible at the target site. The target site can be the site of the bone to be treated, e.g. the site of bone injury, bone ischemia, arthritic site. In case of surgery, at the site of surgery. In case of these administration method intrasynovial, intramuscular or intra-articular administration, into the layers of skin, under the skin into the epidermis, into fat pads muscles of various soft tissues, into bone or bone marrow, etc. In this setting, again injection or any other means to introduce the formulation locally is preferred.

The formulations according to the present invention for therapy may form a film or layer on the bone to be treated.

Also implants administered to the target site can be prepared. In case of chronic bone impairments long term release formulations are preferred.

Specifically preferred are slow-release preparations, e.g. in the form of a hydrogel, a semisolid or solid gel or formulations and delivery systems to provide for the long-acting treatment.

Such kind of formulations can be prepared e.g. in the form of a gel, e.g. as described in US5427778A ("Gel formulations containing growth factors and acrylamide polymer") in which aqueous gel formulations or viscous solutions are described for the controlled delivery of growth factors to a wound site.

The pharmaceutical compositions according to the present invention and for use in accordance to the present invention may comprise, in addition to PF-4 other growth factors as well.

In an embodiment, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredients. The precise nature of the carrier or other material may depend on the route of administration. Those of relevant skill in the art are well able to prepare suitable compositions.

The actual amount administered, and rate and time-course of the administration, will depend on the aim of the administration. If systemic administration is made a relatively high, albeit safe dosage may be necessary to achieve an appropriate level of PF-4 at the target site. Prescriptions of treatment, for example decisions on dosage etc., is in general within the responsibility of general practitioners and other medical doctors.

In a further embodiment PF-4 is present in a plasma fraction preparation.

In a broad sense, blood plasma is the liquid component of blood, in which the blood cells are suspended. It makes up about 60% of total blood volume. Plasma is the supernatant fluid obtained when anti-coagulated blood has been centrifuged. Typically the blood is mixed with an appropriate amount of anticoagulant like heparin, oxalate or ethylenediaminetetraacetic acid (EDTA). This preparation should be mixed immediately and thoroughly to avoid clotting. Blood serum is blood plasma without fibrinogen or the other clotting factors. Serum is clearer than plasma because of fewer proteins. Methods for preparation, optimization and validation of serum and plasma samples from human blood are described e.g. by Ammerlaan W et al. [Ammerlaan W. et al. Method validation for preparing serum and plasma samples from human blood for downstream proteomic, metabolomic, and circulating nucleic acid-based applications. Biopreserv Biobank. 2014 Aug;12(4):269-80.].

As shown in table 1, the level of PF-4 is surprisingly high in plasma and in serum fraction of platelet rich fibrin (SP F). Thus, these preparations are also suitable to administer PF-4. In an embodiment these preparations are also enriched in PF-4.

The serum fraction can be administered alone, or in combination or conjunction with either another agent or any other therapeutic treatment used in the indication, e.g. used to treat patients suffering from osteoarthritis, osteoarthrosis, bone necrosis, or bone ischemia or other diseases disclosed herein. The serum fraction can be administered prior to the administration of the other agent, simultaneously with the agent, or after the administration of the agent. An alternative delivery system provide for the serum fraction associated with or bound to a carrier material, e.g. a gel or an implant.

Table 1 - relative levels of PF-4 in serum fractions

N=3, duplicates

SPRF 2015 vs SERUM 2015

Protein ID SPRF_2015 SERUM_2015 Difference

Refence Spots 86,56 77,13 -9,433 Reference Spots 102,9 100,2 -2,715

PF4 41,87 14,59 -27,28

SPRF 2015 vs PLASMA 2015

Protein ID SPRF_2015 PLASMA_2015 Difference

Refence Spots 86,56 90,07 3,507

Reference Spots 102,9 103,6 0,6730

PF4 41,87 68,16 26,29

SPRF 2015 s PRP 2015

Protein ID SPRF_2015 PRP_2015 Difference

Refence Spots 86,56 64,23 -22,33

Reference Spots 102,9 82,48 -20,46

PF4 41,87 3,109 -38,76

SERUM 2015 vs PLASMA 2015

Protein ID SERUM_2015 PLASMA 2015 Difference

Refence Spots 77,13 90,07 12,94

Reference Spots 100,2 103,6 3,388

PF4 14,59 68,16 53,57

SERUM 2015 s PRP 2015

Protein ID SERUM_2015 PRP_2015 Difference

Refence Spots 77,13 64,23 -12,89

Reference Spots 100,2 82,48 -17,74

PF4 14,59 3,109 -11,48

PLASMA 2015 vs PRP 2015

Protein ID PLASMA_2015 PRP_2015 Difference

Refence Spots 90,07 64,23 -25,83

Reference Spots 103,6 82,48 -21,13

PF4 68,16 3,109 -65,05

Plasma preparation and fractionation kits and protocols are available among many other sources from Thermofisher (3175 Staley Road Grand Island, NY 14072 USA) or Prol mmune (4281 Express Lane,

Suite L2378, Sarasota, FL 34238, USA).

Essentially, a basic plasma preparation can be made as follows: 1. Draw blood into vacutainer tube(s) K₂EDTA (may vary depending on manufacturer). Ensure the correct blood-to-anticoagulant ratio.

2. Invert vacutainer tubes carefully several times to mix blood and anticoagulant and store at room temperature until centrifugation.

3. Samples should undergo centrifugation immediately. This should be carried out for an appropriate time and rotation speed at room temperature (refer to speeds and times recommended by manufacturer). Stop the centrifuge gently (do not brake).

4. This will give three layers: (from top to bottom) plasma, leucocytes (buffy coat), erythrocytes.

5. Carefully aspirate the supernatant (plasma) at room temperature and pool in a centrifuge tube. Take care not to disrupt the cell layer or transfer any cells.

6. Avoid turbidity. Turbid samples should be centrifuged and aspirated again.

7. Store at -80 °C or follow the instruction of your manufactured to include additives.

SPRF can be prepared e.g. as described in Example 1.

The term "platelet rich plasma" or PRP is herein understood as a volume of plasma that has a platelet concentration above baseline. Normal platelet counts in blood range between 150,000/microliter and 350,000/microliter. The platelet concentration is specifically increased by centrifugation, and/or otherwise fractionation or separation of the red blood cell fraction, e.g. centrifugation of whole blood first by a soft spin such as 8 min at 460 g and the buffy coat is used or further pelleted by a hard spin at higher g values. PRP typically comprises an increased platelet concentration, which is about a 1 .5 - 20 fold increase as compared to venous blood.

Such centrifugation and/or fractionation will separate the red blood cells from blood, and further separate the platelet rich fraction (PRP) including platelets, with or without white blood cells together with a few red blood cells from the platelet poor plasma, from platelet poor plasma (PPP). Upon polymerization of fibrin in plasma or PRP platelet rich fibrin (PRF) is formed. The liquid from the fibrinous gel may be conveniently prepared in an appropriate preparation device suitable for aseptic collection of the blood, preparing the PRP, clotting the PRP (e.g. by actively initiating coagulation or by self-activation), separating this coagel to obtain PRF, and optionally further separating the PRF gel to isolate the serum fraction from the PRF coagel.

The acellular or clear supernatant from the PRF may be isolated, or may be removed before fractionating the PRF to isolate the PRF fluid fraction. Such PRF fluid fraction turned out to contain a high concentration of activated platelet releasate and growth factors contained therein. This serum fraction of PRF, hereinafter also referred to as SPRF (serum of PRF), may include the supernatant of the coagel e.g. obtainable by centrifugation, and/or the fluid fraction obtained from the coagel, e.g. SPRF which essentially consists of the fluid fraction. For example, such serum fraction essentially consisting of the PRF fluid fraction is prepared upon fractionating the PRF to isolate the fluid fraction from PRF, e.g. by separating the solid coagel mainly consisting of fibrin gel and platelets. SPRF contains a natural mix of growth factors, some of which are known, yet it is possible that it contains unidentified molecules that have a biological effect. Therefore, its effect on bone regeneration cannot be attributed to a single factor but rather a mix of those factors present. However, it is of importance to the medical community whether it is possible to achieve a bone regeneration effect with a serum fraction to a similar extent than that of recombinant growth factors.

In recent years recombinant growth factors, especially bone morphogenetic proteins BMP-2 and BMP-7 are gaining acceptance as bone growth promoting agents in orthopedic and spinal surgery. It is argued that the use of single growth factors do not mimic the natural environment where several factors are secreted at once, and thus supraphysiological doses are required from these factors to have a robust effect. In our current results we have showed that SPRF, a natural source of growth factors and cytokines have the same effect as the highest, supraphysiological doses of recombinant growth factors on the regeneration of bone cells after damage. This opens the possibility to use the serum fraction SPRF as a therapeutic tool in bone regeneration and bone formation indications. Furthermore, SPRF has the potential to reach the effectiveness of recombinant factors BMP-2, BMP- 7, PDGF or PF-4.

The model used in the present study is not designed to mimic bone healing under normal conditions, but rather regeneration potential of a damaged tissue. We have observed that serum fractions had no effect on the "healthy" state of the bone explants but in the postischemic period both SPRF and PF-4 are effective. This also supports the idea that the current model, with its limitations as an ex vivo system, resembles degenerative bone tissues. It may be assumed that SPRF and probably other plasma preparations with high PF-4 content are preferred variants of the compositions of the invention. The skilled person is aware of preparing bone implants and bone substitutes. In the present invention PF-4 is added to such bone substitutes during preparation.

For example bone substitutes include polymers that e.g. comprises of polylactic acid, polyglycolic acid or poly(lactic-co-glycolic acid). WO2012094708 discloses a synthetic bone replacement material that can prevent potential infection compared to human derived bone allograft. Still till nowadays, bone is the most convenient grafting material, and according to the review article of Konstantinos Anagnostakos et al. [Anagnostakos et al. International Journal of Biomaterials, Volume 2012, Article ID 538061, 9 pages]. This article describes a type of pharmaceutically active components, mainly focusing on antibiotics. A method and graft as described in WO2014/122631 (Lacerta Technologies Inc) wherein a tissue substitute material for implantation based on a natrium-alginate film is disclosed.

Below the invention is illustrated further by non-limiting examples. Example 1: Preparation of an exemplary serum fraction of the invention (SPRF, serum fraction of platelet rich fibrin) and its characterization

Platelet-rich fibrin was prepared by centrifugation of human blood obtained from venipuncture without anticoagulants for 5 minutes at 3000 rpm (1710 g). A fibrinous gel was removed from the tube and the fluid gently squeezed out of the gel to obtain isolated SPRF, which was added to the stem cell medium in 1:4 ratio.

Example 2: Effect of SPRF on Bone explants after oxygen glucose deprivation

Bone explants and oxygen glucose deprivation (OGD), analysis of cell viability by the methyl-thiazol- tetrasolium (MTT) assay

In this in vitro study, bone samples were obtained from the removed femoral head during total hip replacements for primary osteoarthritis. Femoral heads were obtained from patients suffering from coxarthrosis and undergoing hip replacement surgery, during which the femoral head is extracted in its entirety and discarded as surgical waste. Average 0,004 g weight explants (n=40 pieces/ patient) were harvested from the femoral heads The explants were transported into cell culture conditions at 37°C in Dulbecco's Modified Eagle Medium containing 1 g/l glucose, 5% Penicillin-streptomycin and 10% fetal bovine serum (Stem Cell Medium). After an incubation of 3 days of the femoral heads oxygen-glucose deprivation (OGD) was used to model the poor circulation of the femoral head. At a tissue level OGD models cellular damage and impaired regeneration which is characteristic for degenerative bone diseases such as aseptic necrosis, osteochondrosis, osteoarthrosis, etc. The femoral heads were placed into glucose and amino-acid free medium at an oxygen level of O₂<0,5 mmHg (replaced with N₂ gas) for 7 hours after which the normal cell culture conditions were restored.

For quantitative analysis of cell viability the methyl-thiazol-tetrasolium (MTT) assay was used with the following parameters: lh incubation in MTT solution, lh solubilization in isopropanol, absorbance measures at 570 and 690 nm, corrigated with the dry weight of bones. Assay was carried out at 37°C. In preliminary experiments, incubation was tested for 10 minutes, 1, 2, 5 hours, and solubilization in isopropanol was tested for 10 minutes, 1, 2, 3, 4, 5, 6, 20 hours.

The effect of SPRF in comparison with individual platelet derived growth factors

Bone explants were harvested from the discarded femoral heads from patients undergoing hip replacement. Bone grafts of about 10 mm³ were collected and transferred immediately into Dulbecco's Modified Eagle Medium containing 1 g/l of glucose, l%penicillin-streptomycin, and 10% fetal bovine serum. The explants were cultured in this medium under standard cell culture conditions in 24-well plates. Oxygen-glucose deprivation (OGD) was performed in a Pecon incubation system (Erbach-Bach, Germany) on the third day after explantation. The bone pieces were transferred into stem cell medium lacking glucose and amino acids and the oxygen was flushed with nitrogen to 0,5% 02 level for 7 hours. After completion of OGD the medium was replaced and the explants were cultured in 20% oxygen and 5% C02. Blood fractions were added to the medium in a ratio of 1:4 just before OGD and were refreshed at medium changes. SP F was prepared fresh just before use and never stored or frozen. Recombinant human growth factors Bone Morphogenetic Protein 2 (BMP-2), Bone Morphogenetic Protein 7 (BMP-7), Platelet Derived Growth Factor (PDGF) or Platelet Factor 4 (PF-4) were dissolved in stem cell medium and added to the culture in 5, 50, or 500 ng/ml concentrations.

Cell viability was quantified by the MTT assay. The grafts were incubated in a 1:9 diluted mixture of 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl- 2H-tetrazolium bromide (MTT, #M5655, Sigma) and stem cell medium at 37 °C for 60 min then diluted with isopropanol. Absorbance of the solution was measured by a PowerWaveTM XS spectrophotometer at 570 nm and noise was filtered out by measuring the absorbance at 690 nm. The MTT-assay was performed on the third and sixth days after OGD.

There were only a few living cells on the bone chips on the day of the operation, and these cells were damaged. The samples were obtained from different patients. To get the various bone chips into a similar state, they were incubated in stem cell med ium at 37 °C and 5% C02 for 3 days. Sufficient number of cells were detected on the 3rd day, therefore OGD was started to model the ischemic condition for 7 hours. Explants were treated during OGD and continuously for 6 days after OGD with either SPRF or recombinant growth factors in various concentrations. On the 6th day the medium was changed and cell viability assay was done (Figure 1). Example 3: Analysis of the composition of serum fractions For determination the growth factors and angiogenesis-related proteins in the SPRF and plasma protein levels are assessed by using the Proteome Profiler Human Angiogenesis Array Kit (R&D System, #ARY 007). Adobe Photoshop was used for quantitation of protein expression. For the quantitative determination of platelets and ions in SPRF Sysmex XT 4000i and Beckman Coulter AU5800 are used. Results are reported as mean ± SEM. Statistical significances are determined by t-test or one-way ANOVA with Tukey's post-hoc tests as appropriate with the Graphpad Prism software. Significance values of p<0.05 were considered significant.

Claims

1. A method of treating a patient in need of bone regeneration or bone formation due to the condition of said patient, said method comprising administration of an effective amount of platelet factor 4 (PF-4) to said patient.

2. The method according to claim 1 wherein said patient has a condition wherein bone cells of said patient are of have been exposed to ischemia.

3. The method according to claim 1 wherein the condition is selected from degenerative bone diseases.

4. The method according to claim 1 wherein the condition is selected from bone ischemia, bone disease, bone necrosis, osteoarthrosis and osteoarthritis, bone injury, bone graft or bone fraction.

5. The method according to claim 1 wherein PF-4 is recombinant PF-4 or a recombinant human PF-4.

6. The method according to claim 1 wherein PF-4 is present in composition together with several other growth factors being present in said composition.

7. The method according to claim 6 wherein the composition is a serum fraction composition.

8. The method according to claim 7 wherein the serum fraction composition is a plasma composition.

9. The method according to claim 7 wherein the serum fraction composition is a serum fraction of platelet rich fibrin (SP F) composition.

10. The method according to any of claims 6 to 9 wherein the composition comprises a supraphysiological level of PF-4.

11. The method according to claim 1 wherein PF-4 is present in composition which is an implantable composition.

12. A method of promoting in vitro proliferation of cells said method comprising contacting said cells with PF-4 and incubating said cells for a period of time sufficient to promote cell growth or regeneration, wherein the cells are selected from bone cells, in particular osteocytes, osteoclasts, osteoblasts; and bone marrow derived cells.

13. An in vitro method for using PF-4 as a cell culture additive to prepare a bone cell culture.

14. An in vitro method for using PF-4 to prepare a bone implant, said implant comprising PF-4.