WO2025168104A1

WO2025168104A1 - Pharmaceutical composition for inducing new bone formation and method for enhancing spinal fusion and treating bone defects

Info

Publication number: WO2025168104A1
Application number: PCT/CN2025/076447
Authority: WO
Inventors: Bixia He; Hua Zhu Ke; Muyu LI; Wanling LIANG; Liang Liu
Original assignee: Angitia Biomedicines Ltd
Current assignee: Angitia Biomedicines Ltd
Priority date: 2024-02-08
Filing date: 2025-02-08
Publication date: 2025-08-14
Anticipated expiration: 2026-08-08

Abstract

The present invention provides a pharmaceutical formulation, a pharmaceutical composition, a method for inducing new bone formation and a method for accelerating and enhancing spinal fusion and treating bone defects in a subject. The composition comprises autologous coagulum as the carrier for osteogenic bone morphogenetic protein BMP6, which has no immunogenicity to cause inflammation and ectopic bone formation and presents a higher potency for bone formation and reduction of drug usage.

Description

PHARMACEUTICAL COMPOSITION FOR INDUCING NEW BONE FORMATION AND METHOD FOR ENHANCING SPINAL FUSION AND TREATING BONE DEFECTS

Field of the Invention

The present invention relates to a pharmaceutical formulation, a pharmaceutical composition, a method for inducing new bone formation and a method for enhancing and accelerating spinal fusion and treating bone defects in a subject. In particular aspect, the invention relates to a composition forming as an autologous blood coagulum (ABC) comprising the osteogenic bone morphogenetic protein.

Background

Degenerative disk disease (DDD) is a type of disease based on the basic pathology of lumbar disc degeneration, and the prevalence is at a high level.

The main clinical manifestations of degenerative disk disease include pain, numbness, intermittent claudication, stiffness, limited mobility, and spinal deformity. Among them, low back pain (LBP) is the most common clinical symptom of degenerative disc disease. According to 2019 data from the World Health Organization (WHO) , LBP ranks first in disability-adjusted life years, a global burden of disease indicator, among 369 diseases in 204 countries. The treatment of degenerative disc disease, one of the most important causes of LBP, is particularly important. As the disease progresses and disc degeneration worsens, surgical interventions, including interbody fusion, are the last hope for some patients with degenerative disc disease after conservative treatment is ineffective.

Therefore, degenerative disc disease is a common disease that seriously affects Chinese health, and the disability caused by degenerative disc disease damages the health of the majority of patients, and improving the efficacy of lumbar fusion surgery is of great significance to the clinical prognosis of patients with severe degenerative disc disease.

In the past 20 years, lumbar fusion surgery has undergone tremendous changes: 1) Lumbar posterior interbody fusion surgery, including transforaminal lumbar interbody fusion (TLIF) and posterior lumbar interbody fusion (PLIF) , has replaced anterior approach surgery as the most mainstream surgical method. In particular, the proportion of TLIF surgery increased significantly; 2) Polyetheretherketone (PEEK) fusion device composite local autologous bone particle bone grafting has replaced the traditional large autologous iliac bone grafting as a standard intervertebral fusion method, is no less effective than autologous iliac bone grafting, and avoids complications such as pain at the iliac bone extraction.

However, series products, which are currently the most widely used similar products in the world, have only been approved for anterior lumbar fusion surgery (anterior lumbar interbody fusion [ALIF] /oblique lumbar interbody fusion [OLIF] ) in the United States, and have not been approved for use in TLIF or PLIF posterior lumbar fusion surgery. In addition, the positioning of series products is limited to replacing autologous iliac bone grafting, and there is no evidence that series products can bring more benefit to patients than autologous iliac bone grafting in terms of spinal fusion or postoperative function improvement, there is also no evidence that this kind of products has better clinical efficacy than current mainstream fusion methods. In addition, the safety of traditional bone morphogenetic protein (BMP) products is also worrying. Due to the occurrence of adverse reactions such as severe inflammation at the administration site and symptomatic ectopic ossification during application, the clinical use ofseries products has been subject to various restrictions. These adverse reactions are associated with excessive dosage, local rapid release, and strong immunogenicity of bovine collagen sponge carriers.

The patent (CN110612129B) describes an Autologous Bone Graft Substitute composition (ABGS) that facilitates the successful repair of bone defects and promotes new bone growth necessary for spinal fusion, without subjecting the patient to additional risks, pain, or the limitations associated with autograft surgeries. The ABGS disclosed comprises autologous blood, bone morphogenetic protein (BMP) , and a compression resistant matrix (CRM) , or optionally includes a blood clotting agent. The introduction of the compression resistant matrix is essential for several reasons: firstly, it serves as a biocompatible scaffold that structurally supports and reinforces the clot, improving operability during administration and providing a physical framework for new bone formation. Secondly, it enables the ABGS to release BMP6 at the implantation site in a sustainable and durable profile, thereby exerting osteogenic activity at the local administration site while minimizing ectopic bone formation. Furthermore, the CRM or a combination of the CRM with the blood clotting agent is required to reduce the time necessary for clot solidification, thus ensuring the timely and effective administration of the composition.

More importantly, worldwide, there is a lack of clinical products that can promote early spinal fusion. Early fusion after lumbar spine surgery not only marks the early realization of surgical goals. Early integration facilitates the patient's decision to start and strengthen rehabilitation exercises as soon as possible, thereby further accelerating postoperative functional recovery and allowing patients to return to normal work and life as soon as possible after surgery. How to validate the benefits of products in early fusion is a key focus when designing clinical studies.

Although ABGS demonstrates significant improvements over the series in terms of reducing the dosage of drug administration, minimizing localized rapid drug release, and mitigating immunogenicity, there remains potential for further optimization in the simplification of the operational steps and the further reduction of immunogenicity.

Therefore, there is an urgent clinical need for a new product that can be applied in lumbar posterior fusion surgery, which can promote the improvement of clinical efficacy such as spinal fusion in the early postoperative period.

Summary of the Invention

The invention provides a pharmaceutical formulation and a pharmaceutical composition comprising the pharmaceutical formulation. In this pharmaceutical composition, an autologous blood clot was utilized as a delivery carrier for bone morphogenetic protein 6 (BMP6) . Through the optimization of the ratio between autologous blood and BMP-6, and the optimization of the method for preparing the drug before administration, it was unexpectedly found that mixing the BMP6 drug with autologous blood, without the need for any additional components, allows the BMP6-containing autologous blood clot to solidify properly. Furthermore, this configuration enables the BMP6-containing autologous blood clot to release BMP6 at the implantation site in a sustainable and durable manner, thereby exerting osteogenic activity at the local administration site while avoiding ectopic bone formation.

In one aspect, the invention provides a pharmaceutical composition comprising:

(a) autologous blood; and

(b) a pharmaceutical formulation comprising an osteogenic bone morphogenetic protein BMP6 and/or analogs thereof;

wherein the autologous blood mixed with the pharmaceutical formulation forms an autologous blood coagulum (ABC) comprising the osteogenic bone morphogenetic protein.

Some embodiments include the pharmaceutical composition wherein the osteogenic bone morphogenetic protein BMP6 is dimeric recombinant human BMP6 protein, preferably consisting of the amino acid sequence of SEQ ID NO: 1.

In one aspect, the invention provides a pharmaceutical composition comprising:

(a) autologous blood; and

(b) a pharmaceutical formulation comprising osteogenic bone morphogenetic protein BMP6 and/or analogs thereof;

wherein the osteogenic bone morphogenetic protein BMP6 and/or analogs thereof are produced by recombinant DNA technology and expressed in Chinese hamster ovary (CHO) cells;

Some embodiments include the pharmaceutical composition wherein the recombinant DNA technology comprises the step of introducing into CHO cells a nucleic acid molecule encoding the amino acid sequence of SEQ ID NO: 1.

Some embodiments include the pharmaceutical composition wherein the pharmaceutical formulation further comprises a pharmaceutically acceptable adjuvant, a diluent, a carrier, a buffer, a tonicity agent, a chelator, a viscosity modifier, and/or a pH adjusting agent, wherein the pH adjusting agent is selected from the group consisting of: tris buffer, phosphate, acetate, lactate, tris (hydroxymethyl) aminomethane, meglumine (N-methylglucosamine) , citrate, sodium hydroxide, hydrochloric acid, TFA, and amino acids.

Some embodiments include the pharmaceutical composition wherein the pharmaceutical formulation is lyophilized or reconstituted in the form of liquid.

Some embodiments include the pharmaceutical composition wherein the pharmaceutical formulation is prepared in 0.1-1 mg BMP6 and/or analogs thereof per dose, preferably 0.25-0.8 mg BMP6 and/or analogs thereof per dose, more preferably 0.5 mg BMP6 and/or analogs thereof per dose.

Some embodiments include the pharmaceutical composition wherein the pharmaceutical formulation is reconstituted using 0.2-0.4 mL sterile water or water for injection, preferably 0.25mL.

Some embodiments include the pharmaceutical composition wherein a volume ratio between the reconstituted pharmaceutical formulation and the autologous blood is from 1: 7.5 to 1: 12.

Some embodiments include the pharmaceutical composition wherein the volume ratio between the reconstituted pharmaceutical formulation and the autologous blood is selected from 1: 7.5, 1: 8.7, 1: 9.6 or 1: 12.

Some embodiments include the pharmaceutical composition wherein 0.2 to 0.28 mg of the BMP6 and/or analogs thereof per mL of autologous blood is present.

In one aspect, the invention provides a pharmaceutical composition prepared by a method comprising steps of:

(a) mixing the pharmaceutical formulation according to any one of above aspects with autologous blood collected; and

(b) incubating components of step (a) for sufficient time to form an autologous blood coagulum (ABC) .

Some embodiments include the pharmaceutical composition wherein the method further comprising the step of: implanting autologous bone.

Some embodiments include the pharmaceutical composition wherein the method further comprising the step of: implanting autologous bone, allograft bone, xenograft bone, artificial bone, or combinations thereof.

Some embodiments include the pharmaceutical composition wherein the artificial bone is selected from: calcium phosphate ceramics such as tricalcium phosphate (TCP) , hydroxyapatite (HA) , and biphasic calcium phosphate ceramic (containing both TCP and HA in various ratios) ; synthetic polymers (such as polylactic acid, polyglycolic acid, and their copolymers) ; and composite materials (such as combinations of calcium phosphates with collagen) ..

Some embodiments include the pharmaceutical composition wherein the method further comprising the step of: implanting an interbody fusion device.

Some embodiments include the pharmaceutical composition wherein the method comprising reconstituting the pharmaceutical formulation in sterile water or water for injection before step (a) .

Some embodiments include the pharmaceutical composition wherein in step (b) , incubating components of step (a) for 45-180 min, preferably 60-120 min, more preferably 90 min ± 5 min.

Some embodiments include the pharmaceutical composition wherein in step (a) , per mL of autologous blood is mixed with the pharmaceutical formulation comprising 0.2 to 0.28 mg BMP6 and/or analogs thereof.

In one aspect, the invention provides a therapeutic mixture comprising the pharmaceutical composition according to any one of above aspects, wherein the therapeutic mixture further comprising:

autologous bone, allograft bone, xenograft bone, artificial bone, or combinations thereof; and/or an interbody fusion device.

In one aspect, the invention provides a pharmaceutical composition or a therapeutic mixture for use in treating bone-related disorder, inducing new bone formation, promoting bone growth and/or facilitating procedures for generating or restoring bone at a particular site in an individual in need of treatment thereof.

In one aspect, the invention provides a pharmaceutical composition or a therapeutic mixture for use in treating bone defects, degenerative disc disease, spinal non-union or delayed union after spinal fusion surgery, adult scoliosis, trauma (spine reconstruction) , pseudo-arthrosis associated with long bone and spine, tibial non-union fracture, hypophosphatasia, osteogenesis imperfecta, neurofibromatosis type I, atypical osteoporotic fractures, osteoporotic fractures, atypical femoral fracture, vertebral bone fracture, distal radial fracture, facial and cranial injuries or defects, knee osteoarthritis, periodontal disease, teeth and alveolar bone defects and/or alveolar ridge defects.

In one aspect, the invention provides a pharmaceutical composition or a therapeutic mixture for use in spinal fusion surgery, maxilla-cranial reconstruction, high tibial osteotomy, periodontal repair, dental/bone implants, and/or alveolar-ridge augmentation.

In one aspect, the invention provides a pharmaceutical composition or a therapeutic mixture for use in enhancing spinal fusion rate and accelerating spinal fusion after surgeries for treating Degenerative Disc Disease.

Some embodiments include the pharmaceutical composition or the therapeutic mixture for use wherein the surgeries comprising lumbar interbody fusion (LIF) , posterolateral lumbar interbody fusion (PLIF) , anterior lumbar interbody fusion (ALIF) , oblique lumbar interbody fusion (OLIF) , extreme lateral lumbar interbody fusion (XLIF) , direct lateral interbody fusion (DLIF) , and/or open transforaminal lumbar interbody fusion (TLIF) .

Some embodiments include the pharmaceutical composition or the therapeutic mixture for use wherein the surgeries are open transforaminal lumbar interbody fusion (TLIF) .

In one aspect, the invention provides use of the pharmaceutical composition or the therapeutic mixture in the manufacture of a medicament for the treatment of bone-related disorder, inducing new bone formation, promoting bone growth and/or facilitating procedures for generating or restoring bone at a particular site in an individual in need of treatment thereof.

Some embodiments include use of the pharmaceutical composition or the therapeutic mixture wherein the bone-related disorder selected from bone defects, degenerative disc disease, spinal non-union or delayed union after spinal fusion surgery, adult scoliosis, trauma (spine reconstruction) , pseudo-arthrosis associated with long bone and spine, tibial non-union fracture, hypophosphatasia, osteogenesis imperfecta, neurofibromatosis type I, atypical osteoporotic fractures, osteoporotic fractures, atypical femoral fracture, vertebral bone fracture, distal radial fracture, facial and cranial injuries or defects, knee osteoarthritis, periodontal disease, teeth and alveolar bone defects and/or alveolar ridge defects.

Some embodiments include use of the pharmaceutical composition or the therapeutic mixture wherein the treatment of the bone-related disorder selected from spinal fusion surgery, maxilla-cranial reconstruction, high tibial osteotomy, periodontal repair, dental/bone implants, and/or alveolar-ridge augmentation.

Some embodiments include use of the pharmaceutical composition or the therapeutic mixture wherein the treatment of the bone-related disorder selected from enhancing spinal fusion rate and accelerating spinal fusion after surgeries for treating Degenerative Disc Disease.

Some embodiments include use of the pharmaceutical composition or the therapeutic mixture wherein the surgeries comprising lumbar interbody fusion (LIF) , posterolateral lumbar interbody fusion (PLIF) , anterior lumbar interbody fusion (ALIF) , oblique lumbar interbody fusion (OLIF) , extreme lateral lumbar interbody fusion (XLIF) , direct lateral interbody fusion (DLIF) , and/or open transforaminal lumbar interbody fusion (TLIF) .

Some embodiments include use of the pharmaceutical composition or the therapeutic mixture wherein the surgeries are open transforaminal lumbar interbody fusion (TLIF) .

In one aspect, the invention provides a method for treating bone-related disorder, inducing new bone formation, promoting bone growth and/or facilitating procedures for generating or restoring bone at a particular site in an individual in need of treatment thereof wherein the method comprising steps of:

administrating the pharmaceutical composition or the therapeutic mixture according to any one of above aspects to the particular site in an individual in need of treatment for the bone-related disorder.

Some embodiments include the method further comprising the step of:

implanting an autologous bone.

Some embodiments include the method further comprising the step of:

implanting an autologous bone, an allograft bone, a xenograft bone, an artificial bone, or combinations thereof.

Some embodiments include the method further comprising the step of:

implanting an interbody fusion device.

Some embodiments include the method wherein the particular site is the cleared intervertebral space.

Some embodiments include the method wherein the bone-related disorder selected from bone defects, degenerative disc disease, spinal non-union or delayed union after spinal fusion surgery, adult scoliosis, trauma (spine reconstruction) , pseudo-arthrosis associated with long bone and spine, tibial non-union fracture, hypophosphatasia, osteogenesis imperfecta, neurofibromatosis type I, atypical osteoporotic fractures, osteoporotic fractures, atypical femoral fracture, vertebral bone fracture, distal radial fracture, facial and cranial injuries or defects, knee osteoarthritis, periodontal disease, teeth and alveolar bone defects and/or alveolar ridge defects.

Some embodiments include the method wherein the treatment of the bone-related disorder selected from spinal fusion surgery, maxilla-cranial reconstruction, high tibial osteotomy, periodontal repair, dental/bone implants, and/or alveolar-ridge augmentation.

Some embodiments include the method wherein the treatment of the bone-related disorder selected from enhancing spinal fusion rate and accelerating spinal fusion after surgeries for treating Degenerative Disc Disease.

Some embodiments include the method the surgeries comprising lumbar interbody fusion (LIF) , posterolateral lumbar interbody fusion (PLIF) , anterior lumbar interbody fusion (ALIF) , oblique lumbar interbody fusion (OLIF) , extreme lateral lumbar interbody fusion (XLIF) , direct lateral interbody fusion (DLIF) , and/or open transforaminal lumbar interbody fusion (TLIF) .

Some embodiments include the method wherein the surgeries are open transforaminal lumbar interbody fusion (TLIF) .

The pharmaceutical composition and the therapeutic mixture provided by the present invention have achieved favorable results in terms of efficacy and safety in clinical trials, which indicated it’s a promising pharmaceutical composition to be launched for using in the treatment of Degenerative Disc Disease by enhancing and accelerating spinal fusion, promoting bone growth and/or inducing in procedures for generating or restoring bone at a particular site an individual in need.

From the perspective of efficacy, in a randomized, double-blinded, placebo-controlled, phase I/II study to evaluate the safety and preliminary efficacy of single administration of the pharmaceutical composition for lumbar interbody fusion in patients with degenerative disc disease, all major efficacy endpoints are trending in the right direction. The fusion success rates of the pharmaceutical composition treatment groups are numerically higher compared to Placebo group at early stage after the surgery. There was a trend towards earlier successful post-surgical radiographic fusion in the pharmaceutical composition treatment groups than in the placebo group both at month 3 and month 6. These data represent proof of concept that pharmaceutical composition accelerate spinal fusion by promoting new bone formation. The mean reduction from baseline in ODI total score and VAS in the pharmaceutical composition treatment groups are numerically greater than Placebo at various time points, which demonstrated the improvement of clinical outcomes after treatment of the pharmaceutical compositions. The selection of the two time points of "radiographic fusion success rate" at 12 weeks (3 months) after surgery and at 26 weeks (3 months) after surgery are mainly based on the time of BMP to promote bone healing pharmacologically. The early post-surgical period (within 6 months) is the time window in which the pharmaceutical compositions mainly play a role in enhancing and accelerating spinal fusion. The clinical study also showed that the 0.5 mg group of pharmaceutical compositions had a promising effect on accelerating spinal fusion in the early post-surgical period. Achieving successful radiographic fusion at 26 weeks postoperatively reflects the objective clinical benefit of participants in early stage postoperatively. At 26 weeks after surgery, intervertebral bone healing and bone remodeling are relatively mature, and the imaging performance is stable and reliable. Moreover, successful radiographic (bony) fusion is the surgical purpose of intervertebral fusion surgery, and successful fusion marks the realization of surgical goals; Early radiographic fusion helps to decide to let the patients start and intensify rehabilitation exercises as early as possible, helping to improve postoperative recovery.

From the perspective of safety, the pharmaceutical composition was safe and well-tolerated at both dose levels. The incidence of AEs (adverse events) , severe AEs, and drug related AEs in the composition combined group were comparable with the placebo group. Lower rates of serious adverse events (SAEs) and severe AEs were reported in composition 0.5mg dose cohort when compared to placebo group. Overall, the observed adverse events data were as expected in patients with degenerative disc disease being treated with surgical fusion of the lumbar spine and consistent with background comorbidities. Rather than using the bovine Absorbable Collagen Sponge (ACS) as the carrier (e.g. INFUSE) , the pharmaceutical composition using ABC as a non-immunogenic carrier have prevented patients undergoing open transforaminal lumbar interbody fusion surgery from adverse reactions including local inflammation, immunogenicity, heterotopic ossification, and early osteolysis following long bone implantation and spinal fusion. These adverse reactions are mainly caused by the immunogenicity effect of ACS and the burst and uncontrolled release of the active component rhBMP2 from INFUSE when using ACS.

The pharmaceutical formulations with a proper pH of 3.0-5.0 provided by the present invention have superior stability in lyophilized form and are easy to store. At the same time, the pharmaceutical compositions of the present invention, in which autologous blood mixed with the pharmaceutical formulation forms an autologous blood coagulum (ABC) comprising the osteogenic bone morphogenetic protein, have shown satisfactory sustained release in subjects (the rhBMP6 drug slowly released about 20%from ABC in 24 hours, and the sustained release lasts around 15 days) . The sustained release is attributed to the fact that rhBMP6 reversibly binds to plasma protein in autologous blood, and will be gradually released from the coagulum when being administered at the particular local site, especially when being administered to patients undergoing open transforaminal lumbar interbody fusion at site of the intervertebral discs lying between the vertebral bodies.

The contributing factors for the AEs mentioned above include type of carrier used and release kinetics of the BMPs, proinflammatory and angiogenic activity of BMPs, surgical procedure applied, and the dosage administered. The local excessive concentration of BMP2 is generally believed to be the most important contributory factor. BMP2 is delivered using an absorbable bovine collagen sponge (ACS) as carrier. The drawbacks of the ACS are the mechanical weakness and immunogenicity. Mechanically weak ACS are easily compressed by the surrounding tissue or during manual manipulation of the ACS into the cage and cause rapid leakage (burst release) of the BMP2. Burst release is more likely to produce AEs such as concentration-dependent inflammation reactions, edema, pain, and nerve roots irritation. Postoperative nerve-related complications (i.e., neurologic events, back pain, leg pain, radiculitis, and functional loss) and ectopic bone formation can be induced if the leakage occurred with physical proximity to the neural structure or outside the implant site.

The pharmaceutical formulation is delivered using autologous blood coagulum (ABC) , in which rhBMP6 binds tightly to ABC allowing a sustained, stable release of rhBMP6 which attenuates the spread of the inflammatory response to the surroundings in the early stage of the bone formation process. This sustained release kinetics is expected to reduce the AEs such as edema, pain, neurological events stemmed from large inflammatory response and the potential for ectopic bone formation due to ‘burst leakage’ .

In addition, compared to the aforementioned ABGS, the pharmaceutical composition can be simplified by reducing components such as the compressible matrix (CRM) and/or blood clotting agents, thereby reducing the number of steps and the frequency of drug mixing. The active ingredient rhBMP6 reversibly binds to plasma protein in autologous blood in the pharmaceutical composition, allowing for sustainable and durable release from the coagulum when being administered at the particular local site, the following additional beneficial effects have been achieved: 1. Reduction of immunogenic risk caused by the introduction of exogenous substances; decreased opportunity for exposure to non-sterile environments, thereby reducing the risk of surgical site infections; 2. Acceleration of surgical preparation time, facilitating intraoperative handling; 3. Reduction in composition complexity and the number of pre-administration steps, improving the accuracy of drug dosing and helping patients receive the correct drug therapy; 4. Reduction in the workload of medical personnel, improving operational efficiency; 5. Reduction of the components of the pharmaceutical composition, thereby reducing costs.

Brief Description of the Drawings

FIG. 1 shows the physical properties of the rhBMP6/ABC.

FIG. 2A shows the rhBMP6 drug release concentration profiles during 360 h of incubation at room temperature.

FIG. 2B shows the rhBMP6 drug release concentration profiles (semi-log) during 360 h of incubation at room temperature.

FIG. 3 shows the fusion success rate by treatment using the rhBMP6 drug.

FIG. 4 shows the Oswestry Disability Index (ODI) change from baseline.

FIG. 5 shows the Visual Analog Scale (VAS) Score change from baseline.

FIG. 6 shows the efficacy of rhBMP6 drug 0.5 mg treatment in improving the overall success rate.

Detailed Description of the Invention

I. Definitions

In order that the invention may be more clearly understood, the following terms are defined.

As used herein and unless otherwise specified, the term “about” or “approximately” means within plus or minus 10%of a given value or range. Where an integer is required, the term refers to rounding up or down to the nearest integer within plus or minus 10%of the given value or range.

The terms “bone morphogenetic protein” , “BMP” , “osteogenic BMP” , and “morphogen” are synonymous and refer to any member of a particular subclass (i.e., the BMP family) of the transforming growth factor-D (TGF-D) super family of proteins. All such BMPs have a signal peptide, pro-domain, and a carboxy-terminal (mature) domain. The carboxyl-terminal domain is the mature form of the BMP monomer and contains a highly conserved region characterized by seven cysteines, called “7-Cysteine Domain” a hall mark of BMP-Family proteins that form a cysteine knot. BMPs were originally isolated from mammalian bone using protein purification methods. However, BMPs have also been detected in or isolated from other mammalian tissues and organs including kidney, liver, lung, brain, muscle, teeth, and gut. BMPs may also be produced using standard in vitro recombinant DNA technology for expression in prokaryotic-or eukaryotic cell cultures. Some BMPs are commercially available for local use as well.

The term “analogs of BMP6” refer to dimers, heterodimers, truncated forms, or mixtures thereof, which are generated through the expression of BMP6 in prokaryotic or eukaryotic cell cultures using in vitro recombinant DNA technology. The dimers are specifically defined as dimeric recombinant human proteins, typically consisting of two identical protein subunits that are biologically active. The heterodimers are recombinant proteins consisting of two distinct protein subunits with minor variations in their amino acid sequences, which arise during the expression process but retain the biological activity of the native proteins. The truncated forms may include proteins that, while shorter than the full-length protein, retain biologically relevant activity.

BMPs normally exist as dimers of the same monomeric polypeptides (homodimers) held together by hydrophobic interactions and at least one inter-chain (between monomers) disulfide bond. BMPs useful in the compositions and methods described herein are those that have osteogenic activity, i.e., the ability to stimulate bone formation. Osteogenic (or "osteoinductive" ) activity may be detected using any of a variety of standard assays. Such osteogenic assays include ectopic bone formation assays in which a carrier matrix comprising collagen and a BMP is implanted at an ectopic site in a rodent and then monitored for bone formation. In a variation of such an assay, the matrix may be implanted at an ectopic site and the BMP administered to the site, e.g., by intravenous injection into the rodent. Another way to assay for BMP osteogenic activity is to incubate cultured mesenchymal progenitor cells with a BMP and then monitor the cells for differentiation into chondrocytes and/or osteoblasts. BMPs that have osteogenic activity and that are therefore useful in the compositions and methods described herein include, but are not limited to, BMP2, BMP4, BMP6, BMP7, BMP9, BMP12, BMP13, and heterodimers thereof, whether purified from a natural source if any, produced recombinant by eukaryotic (e.g., mammalian, yeasts, insects, fish) or prokaryotic (e.g., bacterial) cells, or produced in whole or in part by in vitro protein synthesis methods. A BMP that has an osteogenic activity may also possess one or more other beneficial pharmacological activities, such as the ability to restore or regenerate damaged soft tissues or organs, e.g., ischemic kidneys.

The term "pharmaceutically acceptable" refers to a material that is not biologically, chemically, or in any other way incompatible with body chemistry and metabolisms and also does not adversely affect the desired, effective activity of an osteogenic BMP or any other component in a composition that may be administered to an individual to promote bone growth according to the invention, one or more named elements or steps also describes the corresponding, more limited, composition or method "consisting essentially of" (or "which consists essentially of" ) the same named elements or steps, meaning that the composition or method includes the named essential elements or steps and may also include additional elements or steps that do not materially affect the basic and novel characteristic (s) of the composition or method. It is also understood that any composition or method described herein as "comprising" or "consisting essentially of" one or more named elements or steps also describes the corresponding, more limited, and close-ended composition or method "consisting of" (or "which consists of" ) the named elements or steps to the exclusion of any other unnamed element or step. In any composition or method disclosed herein, known or disclosed equivalents of any named essential element or step may be substituted for that element or step. Unless indicated otherwise, the meaning of other terms is the same as understood and used by persons skilled in the art, including the fields of orthopedic surgeries, medicine, immunology, biochemistry, molecular biology, and tissue regeneration.

The BMP present in an autologous blood coagulum (ABC) described herein promotes new bone growth from progenitor cells that are present in, or migrate into the defect site where the autologous blood coagulum is implanted. Any osteogenic bone morphogenetic protein (BMP) may be used in the compositions and methods described herein, including analogs thereof, dimers, heterodimers, and combinations (mixtures) of two or more BMPs. Preferred osteogenic BMPs useful in an autologous blood coagulum described herein include, without limitation, BMP2, BMP4, BMP5, BMP6, BMP7, BMP9, BMP12, BMP-13, heterodimers thereof, and combinations thereof. Even more preferred for use in an autologous blood coagulum described herein is an osteogenic BMP selected from BMP2, BMP4, BMP6, BMP7, analogs thereof, dimers, heterodimers thereof, and combinations thereof. Most preferably, the BMP used in an autologous blood coagulum described herein is BMP6.

The pharmaceutical composition in form of autologous blood coagulum (ABC) described herein may also be used to promote new bone growth in any of a variety of orthopedic and dental indications including, but not limited to, spinal fusion, high tibial osteotomy, and maxillofacial augmentations. In the composition, BMP is combined with autologous blood coagulum which is then reinforced with a compression resistant matrix to guide the formation of new bone tissue. BMP6 is a preferred BMP as it does not bind avidly to the BMP antagonist, Noggin, which is abundant in bone, and also binds to most of the BMP6 type I and type II receptors (unlike BMP2 and BMP7) for signaling. An Autologous Blood Coagulum (ABC) is a preferred substrate for the delivery of BMP as a number of plasma proteins bind tightly to BMP6 resulting in the sustained and linear release of BMP6 over seven to ten days. Furthermore, autologous blood contains osteoprogenitors cells (mesenchymal stem cells) which can readily respond to BMP6 during the formation of coagulum to initiate new bone formation at the implant site.

The term “pharmaceutical formulation” or “pharmaceutical composition” refers to preparations which are in such form as to permit the biological activity of the active ingredients to be unequivocally effective, and which contain no additional components which are toxic to the subjects to which the formulation is administered.

The term “lyophilized” as used herein in connection with the formulation according to the invention denotes a formulation which is manufactured by freeze-drying methods known in the art per se. The solvent (e.g. water) is removed by freezing followed by sublimation of the ice under vacuum and desorption of residual water at elevated temperature. The lyophilizate usually has a residual moisture of about 0.1 to 5% (w/w) and is present as a powder or a physically stable cake. The lyophilizate is characterized by a fast dissolution after addition of a reconstitution medium.

The term “reconstituted form” as used herein in connection with the formulation according to the invention denotes a formulation which is lyophilized and re-dissolved by addition of reconstitution medium. Suitable reconstitution media comprise but are not limited to water for injection (WFI) , bacteriostatic water for injection (BWFI) , sodium chloride solutions (e.g. 0.9% (w/v) NaCl) , glucose solutions (e.g. 5%glucose) , surfactant-containing solutions (e.g. 0.01%polysorbate 20) , pH-buffered solutions (e.g. phosphate -buffered solutions) .

The formulation according to the invention is physiologically well tolerated, can be prepared easily, can be dispensed precisely and is stable with respect to decomposition products and aggregates over the duration of storage, during repeated freezing and thawing cycles and mechanical stress.

A “stable” formulation is one in which the protein therein, e.g. the osteogenic bone morphogenetic protein, essentially retains its physical and chemical stability and thus its biological activity upon storage.

The terms “buffer” or “buffering agent” as used herein denotes a pharmaceutically acceptable excipient, which stabilizes the pH of a pharmaceutical preparation. Suitable buffers are well known in the art and can be found in the literature. Independently from the buffer used, the pH can be adjusted to a value in the range from 4.5 to 7.0 and particularly to a value in the range from 5.0 to 6.0 and most particularly to pH 6.0 ± 0.03 with an acid or a base known in the art, e.g. hydrochloric acid, acetic acid, phosphoric acid, sulfuric acid and citric acid, sodium hydroxide and potassium hydroxide, tris buffer, phosphate, acetate, lactate, tris (hydroxymethyl) aminomethane, meglumine (N-methylglucosamine) , citrate, sodium hydroxide, hydrochloric acid, TFA, and amino acids.

A formulation of the present invention can be administered by a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results.

The excipients can also be added as solids to the starting solution comprising the osteogenic bone morphogenetic protein. If the osteogenic bone morphogenetic protein is in the form of a solid, e.g. a lyophilizate, the formulation according to the invention can be prepared by firstly dissolving the osteogenic bone morphogenetic protein in water or buffer solution, optionally comprising one or more of the excipients, and subsequently adding the further excipients as stock solutions or solids. The osteogenic bone morphogenetic protein can advantageously also be dissolved directly in a solution comprising all further excipients. One or more of the excipients present in the formulation according to the invention may already be added during or at the end of the process for the preparation of the osteogenic bone morphogenetic protein, e.g. by dissolving the osteogenic bone morphogenetic protein directly in a solution comprising one, more than one, or preferably all of the excipients of the formulation in the final step of the purification carried out after the preparation of the osteogenic bone morphogenetic protein. If the solution comprising the osteogenic bone morphogenetic protein and the excipients does not yet have the desired pH, this is adjusted by addition of an acid or base, preferably using the acid or base already present in the buffer system. This is followed by sterile filtration.

The stable liquid pharmaceutical formulations according to the invention can also be in a lyophilized form or in a liquid form reconstituted from the lyophilized form. The “lyophilized form” is manufactured by freeze-drying methods known in the art. The lyophilizate usually has a residual moisture content of about 0.1 to 5% (w/w) and is present as a powder or a physically stable cake.

The terms “patient” or “subject” refers to a mammal, such as a human.

The term “autologous bone” , “allograft bone” , “xenograft bone” or “artificial bone” used herein refers to a bone to be placed inside the body of a subject. The bone may have been obtained from a donor, and may have been processed. Where the subject is the donor, the bone is referred to herein as an autologous bone; where the subject is not the donor, the bone graft is referred to herein as an allograft bone. Xenograft bone is derived from animal sources, such as bovine bone. They are processed to remove organic material and reduce the risk of immune reaction. Artificial bone is a biomaterial designed to mimic the function of human bones, the artificial bone used herein is referred to but not limited to biomaterials selected from: calcium phosphate ceramics such as tricalcium phosphate (TCP) , hydroxyapatite (HA) , and biphasic calcium phosphate ceramic (containing both TCP and HA in various ratios) ; synthetic polymers (such as polylactic acid, polyglycolic acid, and their copolymers) ; and composite materials (such as combinations of calcium phosphates with collagen) ..

The terms “disorder” and “disease” are synonymous and refer to any pathological condition, irrespective of cause or etiological agent. A “defect” in a bone or other tissue refers to a site of abnormal or deficient tissue growth. A “disease” or “disorder” may be characterized by one or more “defects” in one or more tissues. As used herein, the terms “treatment” and “treating” refer to any regimen that alleviates one or more symptoms or manifestations of a disease or disorder, that inhibits, arrests or reverses (causes regression) of a disease or disorder, or that prevents onsets of a disease or disorder. The term “treatment” includes prophylaxis (prevention) of one or more symptoms or manifestations of a disease, including ameliorating or inhibiting the extent of a symptom or manifestation, including pain, that would otherwise characterize the disease in the absence of the treatment.

A composition or method described herein as “comprising” one or more named elements or steps is open-ended, meaning that the named elements or steps are essential, but other elements or steps may be added within the scope of the composition or method. To avoid prolixity, it is also understood that any composition or method described herein as “comprising” (or “which comprises” ) .

II. Detailed Description

In one aspect, the invention provides a pharmaceutical composition comprising:

(a) autologous blood; and

In one aspect, the invention provides a pharmaceutical composition comprising:

(a) autologous blood; and

Some embodiments include the pharmaceutical composition wherein the pharmaceutical formulation is lyophilized.

Some embodiments include the pharmaceutical composition wherein the pharmaceutical formulation is in reconstituted the form of liquid.

Some embodiments include the pharmaceutical composition wherein 0.1-1 mg BMP6 and/or analogs thereof of the pharmaceutical formulation is present, such as 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95mg.

Some embodiments include the pharmaceutical composition wherein 0.25-0.8 mg BMP6 and/or analogs thereof of the pharmaceutical formulation is present.

Some embodiments include the pharmaceutical composition wherein 0.5mg BMP6 and/or analogs thereof of the pharmaceutical formulation is present.

Some embodiments include the pharmaceutical composition wherein the pharmaceutical formulation is reconstituted using 0.2-0.4 mL sterile water or water for injection, such as 0.2, 0.25, 0.3, 0.35, 0.4 mL.

Some embodiments include the pharmaceutical composition wherein the pharmaceutical formulation is reconstituted using 0.25mL sterile water or water for injection.

Some embodiments include the pharmaceutical composition wherein a volume ratio between the pharmaceutical formulation and the autologous blood is from 1: 7.5 to 1: 12.

Some embodiments include the pharmaceutical composition wherein the volume ratio between the pharmaceutical formulation and the autologous blood is selected from 1: 7.5, 1: 8.7, 1: 9.6 or 1: 12.

Some embodiments include the pharmaceutical composition wherein 0.2 to 0.28 mg BMP6 and/or analogs thereof of the pharmaceutical formulation per mL of autologous blood is present.

Some embodiments include the pharmaceutical composition wherein the method further comprising the step of: implanting autologous bone, allograft bone, a xenograft bone, artificial bone, or combinations thereof.

Some embodiments include the pharmaceutical composition wherein the method further comprising the step of: implanting an interbody fusion device after step.

In one aspect, the invention provides a therapeutic mixture comprising the pharmaceutical composition according to any one of claims 1-15, wherein the therapeutic mixture further comprising:

Some embodiments include the method further comprising the step of:

implanting an autologous bone.

Some embodiments include the method further comprising the step of:

implanting an interbody fusion device.

Example

Example 1: rhBMP6/ABC composition preparation

1.rhBMP6 drug substance preparation

The rhBMP6 drug substance is produced by recombinant DNA technology and expressed in Chinese hamster ovary (CHO) cells. The amino acid sequence of rhBMP6 monomer is shown in SEQ ID NO: 1. The dimeric rhBMP6 protein has a molecular weight of approximately 35 kilodaltons and approximately 17 kilodaltons after reduction to monomers.

The amino acid sequence of rhBMP6 monomer:

The nucleic acid molecule encoding the rhBMP6 monomer is transfected into CHO cells, and rhBMP6 is expressed in the CHO cells. The rhBMP6 is then purified and recovered to obtain a drug substance comprising dimeric rhBMP6 protein.

The final rhBMP6 drug product is formulated and supplied as a lyophilized powder in glass vials. Table 1 shows the composition of a vial of rhBMP6. The rhBMP6 drug product should be stored at 2 to 8℃ or at -20℃ prior to reconstitution with water for injection (WFI) and subsequent mixing with the patient’s blood.

Table 1. Composition of rhBMP6 Drug Product.

Prior to use vial (s) are reconstituted using sterile WFI and the required volume mixed with the patient’s blood according to procedures that are specific for each application (trial indication and design) . Autograft collected during the surgery procedure can be added to the intended fusion site along with rhBMP6/ABC (the ready to use product) to enhance the biomechanical properties.

The physical and biological properties of rhBMP6, after reconstitution with WFI, are shown in Table 2.

Table 2. Physical and Biological Properties of Reconstituted rhBMP6.

2. ABC component

Due to its nature, the ready to use product, rhBMP6/ABC, should be prepared immediately prior to application. Briefly, preparation steps entail: (a) reconstitution of rhBMP6 with WFI and aspiration into a syringe; (b) blood withdrawal; (c) mixing of the components using interconnected syringes; (d) incubation of the syringe with blood containing rhBMP6/at room temperature until the formation rhBMP6/ABC (ready-to-use product) ; (e) quality control of rhBMP6/ABC.

The physical properties of the rhBMP6/ABC, are shown in Table 3 and FIG 1.

Table 3. Physical Properties of the rhBMP6/ABC.

Example 2: Stability of rhBMP6 drug substance in ABC

Fresh whole blood was collected from 6 healthy volunteers, and 6 autologous blood coagulums (ABCs) were prepared for each individual as follows, and serum samples and homogenate samples were collected separately:

Sample 1: 2 mL of fresh whole blood was taken and placed for 90 min ± 5 min, the blank ABCs were removed and weight, the were placed in homogenising tubes, 208 μL of rhBMP6 drug solution was added (containing 0.5 mg of rhBMP6 drug) , and then 2%SDS solution was added (containing enzyme inhibitor) (the volume of which is 5 times the volume of the ABC -208 μL) , homogenise, centrifuge and collect the supernatant homogenate as a time 0 sample.

Samples 2 -6: 2 mL of fresh whole blood was rapidly transferred to the sample tubes (Tubes 2 -6) containing 208 μL of rhBMP6 drug solution (containing 0.5 mg of rhBMP6 drug) , respectively, mixed well, and were placed at room temperature for 30 min ± 2 min, 45 min ± 2 min, 60 min ± 5 min, 90 min ± 5 min, 120 min ± 5 min, 180 min ± 10 min, the state of the coagulums was observed and photographed, and the coagulums were fished out and the upper serum sample layer was collected by centrifuging the original tube at 4000 g for 5 min. The coagulums were weighed, homogenised, centrifuged, and approximately 500 μL of supernatant homogenate was collected (first and backup, approximately 250 μL each) .

Calculation formula:

Homogenate volume (mL) = weight of blood coagulum (g) /ρ*× 6;

Volume of serum (mL) = volume of whole blood (mL) + volume of rhBMP6 drug (mL) -weight of blood coagulum (g) /ρ*;

Amount of drug in blood coagulum# (mg) = (homogenate concentration x homogenate volume) /homogenate recovery;

Amount of drug in serum (mg) = serum concentration x serum volume.

*Assuming a coagulum density (ρ) of 1.0 g/mL.

#The results of the homogenate concentration assay are for reference only as there is no data to support the stability of rhBMP6 drug in homogenate. The following indirect method was used to calculate the amount of drug in the blood coagulum and the percentage of drug in the blood coagulum:

Amount of drug in blood coagulum (mg) = 0.5 mg -amount of drug in serum (mg) ;

Proportion drug in blood coagulum %= amount of drug in blood coagulum (mg) /0.5 mg × 100%. Result:

A total of 30 drug-containing blood coagulums prepared from fresh whole blood of 6 healthy volunteers were left at room temperature for 30 min; all coagulums were fully formed; and no significant change in the morphology of the coagulums was observed during the observation period of 30-180 min.

After the drug-containing blood coagulums were placed at room temperature for 45 min, 60 min, 90 min, 120 min and 180 min, the mean rhBMP6 drug content (range) in the coagulums was 0.46 mg (0.44-0.47 mg) , 0.46 mg (0.45-0.47 mg) , 0.45 mg (0.44-0.46 mg) , 0.45 mg (0.43-0.46 mg) , and 0.43 mg (0.43-0.45 mg) . The results showed that the content of rhBMP6 drug in the coagulums formed after mixing rhBMP6 drug solution (containing 0.5 mg of rhBMP6 drug) and whole blood and standing for different times (45-180 min) was basically the same. The data are detailed shown in Table 4.

Table 4. Drug content in blood coagulums

Example 3: Effect of different (rhBMP6 drug solution: whole blood) volume ratios on drug content in ABC

Fresh whole blood was collected from six healthy volunteers separately, and fresh whole blood of each individual was mixed with rhBMP6 drug solution according to the four volume ratios shown in the Table 5 below, and after placed at room temperature for 90 min ± 5 min, the ABC were fished out, homogenised, and centrifuged, and approximately 500 μL of the supernatant homogenate (the first and the backup, each of which was approximately 250 μL) were collected, and the supernatant sample was collected by centrifugation of the original tubes for 5 min at 4,000 g. The supernatant sample was collected by centrifugation of the original tubes at 4,000 g. The supernatant sample was collected by centrifugation of the original tube at 5 min.

Table 5. different volume ratios of rhBMP6 drug solution: whole blood

*Based on a measured protein content of 0.6 mg in CoA.

Result:

In drug-containing haemoclots prepared from fresh whole blood of six healthy volunteers (rhBMP6 drug solution: whole blood) in volume ratios of 1: 8.7, 1: 9.6, 1: 12, 1: 7.5 (5-6 portions/ratio, each portion was added with 0.5 mg of rhBMP6 drug) , the mean drug content (range) was 0.46 mg (0.44-0.47 mg) , 0.45 mg (0.43-0.46 mg) , 0.44 mg (0.42-0.46 mg) , and 0.45 mg (0.44-0.47 mg) , respectively; and the mean (range) drug content in the blood coagulum as a percentage %of the total amount of drug added was 90.8% (88.1%-93.5%) , 89.2% (86.5%-92.7%) , 87.8% (85.0%-91.4%) , and 90.7% (87.5%-93.6%) . The results showed that the content of rhBMP6 drug in the blood coagulum was basically the same when the blood coagulum was prepared with different (rhBMP6 drug solution : whole blood) volume ratios (1: 7.5 -1: 12) . The data are shown in Table 6.

Table 6. Amount of drug in ABC

Example 4: Release curves of ABC prepared in different (rhBMP6 drug solution : whole blood) volume ratios

Fresh whole blood was collected from six healthy volunteers, and four ABC with different (rhBMP6 drug solution: whole blood) volume ratios were prepared as described in Example 3, weighed, and transported (within 48 h) to the testing laboratory at 2-8℃.

To each coagulum, 2 mL of blank medium was added and incubated at 25℃, with the addition of medium recorded as time 0. Samples of incubation medium (200 μL/sample, 2 samples) were taken at 1 h ± 5 min, 8 h ± 10 min, 24 h ± 15 min, and every 24 h ± 30 min up to 360 h for analysis of rhBMP6 drug concentration; after each sample, the remaining incubation medium was discarded, and 2 mL of blank medium was reintroduced (at least 30 min in advance) at 25℃, and then continue incubation at 25℃.

To further examine the influencing factors (coagulum morphology, coagulum to buffer volume ratio) affecting the release of rhBMP6 drug from the blood coagulum, after 360 h of sampling, 8 blood coagulums from Individual 1 and Individual 2 were chopped and placed in 50 mL centrifuge tubes, and incubated by adding 20 mL of 1*PBS; 8 blood coagulums from Individual 3 and Individual 4 were placed in 50 mL centrifuge tubes, and 20 mL of 1*PBS was added for incubation; 8 blood coagulums from Individual 5 and Individual 6 were weighed individually and approximately 1/10 of the blood coagulums were separated from them and placed in 50 mL centrifuge tubes and 20 mL of 1×PBS was added for incubation. The incubation temperature was 37℃, and fluid changes were performed using 1×PBS at intervals of 0 h, 72 h ± 30 min, and 96 h ± 30 min. At 120 h ± 30 min of incubation, the medium was changed for fluid changes, and 15 mL of medium was added to each sample, and the samples were collected after 24 h ± 30 min (i.e., 504 h samples) . Samples were taken at 200 μL/sample and 2 copies were preserved for rhBMP6 drug concentration analysis.

Result:

The rhBMP6 drug release concentration profiles (see FIG. 2) of the haematoclots prepared in four (rhBMP6 drug solution: whole blood) volume ratios (1: 8.7, 1: 9.6, 1: 12, 1: 7.5) during 360 h of incubation at room temperature in the medium were essentially coincident and did not show any significant differences. The results showed that the rhBMP6 drug solution to whole blood volume ratios were in the range of 1: 7.5 -1: 12, and the in vitro release characteristics of rhBMP6 drug from blood coagulums were similar.

Example 5: Efficacy and Safety Assessments

Indications: Degenerative disc disease requiring single-level interbody fusion of the spine

Study Population: Patients with degenerative disc disease requiring single-level interbody fusion of the spine.

Investigational Drugs, Specification, Dosage Forms and Modes of Administration:

rhBMP6 drug:

Specification: 0.5 mg/vial

Dosage Form: Sterile powder for injection

Mode of Administration: Single intervertebral local administration (intraoperative)

Placebo:

Dosage Form: Sterile powder for injection (excipients only)

Study Duration:

Planned Screening Duration: Up to 14 days

Duration of Planned Treatment: Single dose on the day of surgery

Duration of Planned Randomized Controlled Period: 52 weeks (1-52 weeks)

Duration of Planned Open-label Long-term Safety Follow-up: 52 weeks (53-104 weeks)

Total Study Duration: Up to 106 weeks.

This is a multicenter, randomized, double-blind, placebo-controlled phase 3 clinical study designed to evaluate the efficacy, safety, tolerability, and PK characteristics and immunogenicity of a single intervertebral local administration of rhBMP6 drug in patients with degenerative disc disease undergoing lumbar interbody fusion. The study includes a 2-week screening period, dosing (surgical) days, a 52-week randomized controlled period, and a 53-104-week Open-label Long-term Safety Follow-up period.

The subjects who meet all the inclusion criteria and do not meet any of the exclusion criteria will be randomized to the test or the control group in a 1: 1 ratio and stratified according to whether subjects are over 65 years of age (inclusive) and whether they smoke. They will receive a single local intervertebral dose of rhBMP6 drug 0.5 mg or placebo, respectively. The surgical method is open transforaminal lumbar interbody fusion (TLIF) from posterior approach. The fusion segment during surgery is a single intervertebral segment between the third lumbar vertebrae and the first sacral vertebrae (L3-S1) .

During the preoperative screening period, the subjects need to complete Oswestry disability index (ODI) score, low back/leg pain NRS score, EQ-5D-5L scale score and safety baseline tests.

During the randomized controlled period, the subjects will be followed up at the sites at 5 days, 6, 12, 26, and 52 weeks after the surgery. All subjects will be required to undergo spine X-rays and CT at 5 days, 12, 26, and 52 weeks after surgery to evaluate the spinal fusion status. All subjects need to complete ODI questionnaire, low back pain/leg pain NRS score, EQ-5D-5L scale at 12, 26 and 52 weeks post-surgery. The postoperative recovery questionnaire and healthcare resource utilization questionnaires will be completed at 6, 12, 26, and 52 weeks after surgery. To evaluate radiographic fusion and bone formation, the imaging results at 5 days after surgery will be used as the baseline. X-ray and CT results for efficacy evaluation will be evaluated by an independent central imaging evaluation center. According to the protocol, blood samples will be collected at the planned visits for PK and anti-drug antibody (ADA) , and safety data will be collected postoperatively. AEs including soft tissue ossification at the site of administration and additional surgery will be of special interest during the study.

During the open-label long-term safety follow-up period, subjects will be followed up by telephone 78 weeks post-surgery and 104 weeks post-surgery at the study site for safety assessment.

The results showed that rhBMP6 drug had a good overall safety and tolerability at two doses (0.25 mg or 0.5 mg) . After a single intervertebral administration of rhBMP6 drug with ABC as a carrier, there was no systemic exposure associated with medication and no ADA was produced. rhBMP6 drug has shown preliminary clinical efficacy in promoting spinal fusion, improving function, and pain scores.

Number of Subjects:

Based on the phase I/II clinical study, 81%and 40%of subjects with baseline ODIs greater than or equal to 30 before surgery in the rhBMP6 drug and placebo groups, respectively, achieved overall postoperative success. Of these, 81%and 60%of subjects in the rhBMP6 drug group and placebo group achieved successful fusion at 6 months post-surgery, 100%and 94%achieved successful fusion at 12 months post-surgery, and 100%and 81%achieved successful ODI improvement at 12 months post-surgery. In this study, it is conservatively assumed that approximately 60%of subjects in the rhBMP6 drug group and 40%of subjects in the placebo group will achieve overall postoperative success. At a 2-sided significance level of 5%, using stratified Cochran-Mantel-Haenszel (CMH) test and assuming that 15%of subjects may withdraw from the study, participation of approximately 400 subjects (approximately 200 per group) may result in more than 90%power for hypothesis testing to demonstrate superiority of rhBMP6 drug over placebo. The sample size is calculated using the commercial software EAST (version 6.5.3) .

Efficacy Assessment

Radiographic Fusion Assessment

Imaging results at 5 days postoperatively are used as a baseline. The subjects will receive radiographic evaluation (including CT and X-ray examination) at 12, 26 and 52 weeks post-surgery/at early withdrawal visit of the randomized controlled period.

Radiographic fusion success will be determined by an independent central imaging evaluation center according to the following criteria:

1. Evidence of intervertebral bone fusion as demonstrated by continuous bone bridging from the superior and inferior intervertebral body at the target level based on CT;

2. No more than 3 mm in translation motion at the treated level on lateral flexion/extension radiographs;

3. Less than 5° in angular motion at the treated level on lateral flexion/extension radiographs.

Disability and Pain Assessment

Subjects will be required to complete the ODI questionnaire (Oswestry Disability Index (ODI) Questionnaire) and low back/leg pain NRS scoring before surgery and at 12, 26 and 52 weeks after surgery/at early withdrawal visit of the randomized controlled period to assess the subject's disability and pain relief. The results of the preoperative evaluation collected will be used as a baseline.

Evaluation of Bone Formation

The independent central imaging evaluation center will assess new bone mass in the intervertebral space at the surgical level according to the CT results at 12, 26 and 52 weeks post-surgery/at early withdrawal visit of the randomized controlled period. Imaging results at 5 days post-surgery will be used as baselines for evaluation of bone formation.

Safety Assessment

Safety assessments include the incidence of AEs during the study, their causality with the investigational drug and intensity, the incidence of AESIs (soft tissue ossification at the site of administration and additional surgery) , vital signs, physical examination, safety laboratory tests, ECGs, etc.

PK Evaluation

Blood samples from the first 16 subjects will be collected at the time points specified in the Schedule of Activities (SOA) for PK analysis.

Immunogenicity Assessment

Blood samples will be collected for ADA testing from all subjects at time points specified in the SOA, and serum will be collected to assess potential antibody formation and dynamic changes.

Assessment of Quality of Life, Postoperative Recovery, and Healthcare Resource Utilization:

Subjects will complete the EQ-5D-5L scale, the post-surgery recovery questionnaire, and the healthcare resource utilization questionnaire at the time points specified in the SOA to assess the quality of life, postoperative recovery, and healthcare resource utilization of subjects, respectively.

Statistical Analyses

Efficacy Analysis

Primary Endpoint Analysis

Overall success rate in the primary endpoint is defined as the proportion of subjects who achieve overall success. Overall success is defined as successful radiographic fusion at 26 weeks post-surgery, successful radiographic fusion at 52 weeks post-surgery and successful ODI improvement at 52 weeks post-surgery. The primary efficacy analysis will be conducted on the Full Analysis Set (FAS) . The analysis will be performed by a CMH test on overall success rates of subjects in the rhBMP6 drug group and the placebo group using stratification factors of smoking (yes vs. no) and age group (< 65 vs.≥ 65 years) , and the odds ratio of success rates between groups and 95%confidence interval (CI) will be estimated.

Analysis of Key Secondary Endpoints

Key secondary endpoints include:

1. Success rate of radiographic fusion at 26 weeks post-surgery;

2. Mean change in ODI score from preoperative baseline at 52 weeks post-surgery;

3. Success rate of radiographic fusion at 12 weeks post-surgery;

4. Mean change in ODI score from preoperative baseline at 26 weeks post-surgery.

5. Success rate of radiographic fusion at 52 weeks post-surgery.

The method of analyzing the success rate of spinal fusion at 12, 26 and 52 weeks post-surgery will be consistent with that of the analysis of primary endpoint. The analysis will be performed by a CMH test on the fusion rates of subjects in the rhBMP6 drug group and the placebo group using stratification factors of age group (< 65 vs. ≥ 65 years) and smoking (yes vs. no) , and the odds ratio of fusion rates between groups and 95%CI will be estimated.

In key secondary analyses, the change in ODI from baseline at 26 and 52 weeks post-surgery will be analyzed by mixed-effects models of repeated-measure data (MMRM) . The change in ODI from baseline is the dependent variable of the model. Independent variables of the model include treatment group, visit time, interaction variable of treatment group and visit time, randomization stratification variables (age [< 65 years vs. ≥ 65 years] and smoking [yes vs. no] ) , and baseline value of ODI.

Multiple Sequential Tests

If the superiority hypothesis test for the primary endpoint successfully rejects the null hypothesis, the key secondary endpoints will be tested in the following hierarchical manner.

1. Success rate of radiographic fusion at 26 weeks post-surgery;

3. Success rate of radiographic fusion at 12 weeks post-surgery;

5. Success rate of radiographic fusion at 52 weeks post-surgery

Efficacy: All major efficacy endpoints are trending in the right direction.

The fusion success rates of rhBMP6 drug treatment groups are numerically higher compared with Placebo starting from Month 3. And the treatment difference gets smaller over time as expected. (see FIG. 3)

The medium of new bone formation (%) for rhBMP6 drug treatment groups are numerically greater than Placebo starting from Month 6. New bone formation grade data reflects the same treatment effects.

The mean reduction from baseline in ODI total score and VAS in rhBMP6 drug treatment groups are numerically greater than Placebo at various time points. (see FIG. 4 and FIG. 5)

Safety Analysis

Treatment-emergent adverse events (TEAEs) are defined as AEs that first occur or worsen in severity from the administration of investigational drug up to 52 weeks.

AEs will be coded from actual term using the Medical Dictionary for Regulatory Activities (MedDRA) and reported with preferred terms and system organ class. Summary statistics will be provided for incidence of AEs, TEAEs, SAEs, drug-related AEs, drug-related TEAEs, drug-related SAEs, AEs leading to study discontinuation, deaths and AESIs. The number of events and proportions of subjects experiencing AEs will be reported for each treatment group.

The measured values in laboratory tests, including clinical chemistry, haematology, and urinalysis, and their changes from baseline will be summarized by treatment group and visit. The measured values of systolic and diastolic blood pressure, pulse rate, ECG interval and the change from baseline will be summarized by treatment group and visit.

Safety: rhBMP6 drug was safe and well-tolerated at both dose levels

The incidence of AEs, severe AEs, and drug related AEs in the rhBMP6 drug combined group were comparable with the placebo group.

Lower rates of SAEs and severe AEs were reported in rhBMP6 drug 0.5mg dose cohort when compared to placebo group.

Overall, the observed adverse events data were as expected in patients with degenerative disc disease being treated with surgical fusion of the lumbar spine and consistent with background comorbidities.

There was no new safety signal identified.

Table 7. Overview of Any Adverse Events (AEs) (12 months)

Note: #is the number of events; n (%) = number of study participants reporting at least 1 AE (subject incidence)

1. Any AE/SAE is defined as all adverse events/serious adverse events that happened between the study drug treatment and the end of the trial (12 months) .

2. Drug related AE/SAE is defined as: definitely related to study drug, possible related to study drug, or uncertain

A phase 1/2 clinical study of rhBMP6 drug has currently been completed. The preliminary fusion-promoting effect of rhBMP6 drug was observed in the study. Compared with success rates of radiographic fusion of 21.1%, 60%and 90.5%at 3, 6 and 12 months after surgery in the control group, the fusion success rate was higher in the rhBMP6 drug 0.5 mg treatment group at each time point, which was 45.0%, 80.0%, and 100%at 3, 6, and 12 months after surgery, respectively. At 12 months after surgery, the median proportion of neonatal bone mass in the rhBMP6 drug 0.5 mg treatment group and the control group were 20.55%and 3.47%, respectively. In terms of postoperative functional recovery and pain relief, the ODI scores in the rhBMP6 drug 0.5 mg treatment and control groups at 12 months after surgery were 9.1 and 24.8, respectively, which decreased (improved) from baseline by 36.4 (76.5%) and 24.4 (46.8%) ; the pain scores of Visual Analog Scale (VAS) in the rhBMP6 drug 0.5 mg treatment and control groups were 1.3 and 2.3, respectively, which decreased (improved) from baseline by 3.8 (73.3%) and 2.9 (44.3%) , indicating that the rhBMP6 drug 0.5 mg treatment group was numerically superior to the control group in postoperative functional recovery and pain relief.

Analysis of the phase 1/2 clinical study in patients with preoperative baseline ODI ≥ 30 points (i.e. the severe portion of patients) , showed that 81%of subjects in the rhBMP6 drug 0.5 mg treatment group and 60%in the placebo group achieved successful fusion at 6 months post-surgery, 100%and 94%achieved successful fusion at 12 months post-surgery, and 100%and 81%achieved successful ODI improvement at 12 months post-surgery. The overall success rate of 81.3%in the rhBMP6 drug 0.5 mg treatment was significantly higher than that in the control group of 40.0%. The efficacy of rhBMP6 drug 0.5 mg treatment in improving the overall success rate was verified (see FIG. 6) .

The study showed that a single dose of rhBMP6 drug 0.5 mg was safe and well tolerated in an intervertebral disc fusion surgery. The majority of the AEs were mild to moderate in severity during the study and were judged to be unrelated to the drug. No drug-related SAEs were reported in any of treatment groups of rhBMP6 drug during the study. The types of common AEs in the rhBMP6 drug 0.5 mg treatment group were similar to those in the placebo group and the incidence was similar. There were no AEs suggestive of hypersensitivity, nerve root irritation, or radiculopathy in any treatment group of rhBMP6 drug. No events of ectopic bone formation were identified by either AE reports or radiographic assessments.

There were no identified risks for rhBMP6 drug.

Claims

A pharmaceutical composition comprising:

(a) autologous blood; and

(b) a pharmaceutical formulation comprising osteogenic bone morphogenetic protein BMP6 and/or analogs thereof;

wherein the autologous blood mixed with the pharmaceutical formulation forms an autologous blood coagulum (ABC) comprising the osteogenic bone morphogenetic protein.
The pharmaceutical composition according to claim 1, wherein the osteogenic bone morphogenetic protein BMP6 is dimeric recombinant human BMP6 protein, preferably consisting of the amino acid sequence of SEQ ID NO: 1.
A pharmaceutical composition comprising:

(a) autologous blood; and

(b) a pharmaceutical formulation comprising osteogenic bone morphogenetic protein BMP6 and/or analogs thereof;

wherein the osteogenic bone morphogenetic protein BMP6 and/or analogs thereof are produced by recombinant DNA technology and expressed in Chinese hamster ovary (CHO) cells;

wherein the autologous blood mixed with the pharmaceutical formulation forms an autologous blood coagulum (ABC) comprising the osteogenic bone morphogenetic protein.
The pharmaceutical composition according to claim 3, wherein the recombinant DNA technology comprises the step of introducing into CHO cells a nucleic acid molecule encoding the amino acid sequence of SEQ ID NO: 1.
The pharmaceutical composition according to any one of claims 1-4, wherein the pharmaceutical formulation further comprises a pharmaceutically acceptable adjuvant, a diluent, a carrier, a buffer, a tonicity agent, a chelator, a viscosity modifier, and/or a pH adjusting agent.
The pharmaceutical composition according to any one of claims 1-5, wherein the pharmaceutical formulation is lyophilized or reconstituted in the form of liquid.
The pharmaceutical composition according to claim 6, wherein the pharmaceutical formulation is prepared in 0.1-1 mg BMP6 and/or analogs thereof per dose, preferably 0.25-0.8 mg BMP6 or analogs thereof per dose, more preferably 0.5 mg BMP6 and/or analogs thereof per dose.
The pharmaceutical composition according to claim 6 or 7, wherein the pharmaceutical formulation is reconstituted using 0.2-0.4 mL sterile water or water for injection, preferably 0.25 mL.
The pharmaceutical composition according to any one of claims 6-8, wherein a volume ratio between the reconstituted pharmaceutical formulation and the autologous blood is from 1: 7.5 to 1: 12.
The pharmaceutical composition according to claim 9, wherein the volume ratio between the reconstituted pharmaceutical formulation and the autologous blood is selected from 1: 7.5, 1: 8.7, 1: 9.6 or 1: 12.
The pharmaceutical composition according to any one of claims 1-10, wherein 0.2 to 0.28 mg of the BMP6 and/or analogs thereof per mL of autologous blood is present.
A pharmaceutical composition prepared by a method comprising steps of:

(a) mixing the pharmaceutical formulation according to any one of claims 1-11 with autologous blood collected; and

(b) incubating components of step (a) for sufficient time to form an autologous blood coagulum (ABC) .
The pharmaceutical composition according to claim 12, wherein the method comprising reconstituting the pharmaceutical formulation in sterile water or water for injection before step (a) .
The pharmaceutical composition according to claim 12 or 13, wherein in step (b) , incubating components of step (a) for 45-180 min, preferably 60-120 min, more preferably 90 min ± 5 min.
The pharmaceutical composition according to any one of claims 12-14, wherein in step (a) , per mL of autologous blood is mixed with the pharmaceutical formulation comprising 0.2 to 0.28 mg BMP6 and/or analogs thereof.
A therapeutic mixture comprising the pharmaceutical composition according to any one of claims 1-15, wherein the therapeutic mixture further comprising:

autologous bone, allograft bone, xenograft bone, artificial bone, or combinations thereof; and/or

an interbody fusion device.
The pharmaceutical composition according to any one of claims 1-15 or the therapeutic mixture according to claim 16 for use in treating bone-related disorder, inducing new bone formation, promoting bone growth and/or facilitating procedures for generating or restoring bone at a particular site in an individual in need of treatment thereof.
The pharmaceutical composition according to any one of claims 1-15 or the therapeutic mixture according to claim 16 for use in treating bone defects, degenerative disc disease, spinal non-union or delayed union after spinal fusion surgery, adult scoliosis, trauma (spine reconstruction) , pseudo-arthrosis associated with long bone and spine, tibial non-union fracture, hypophosphatasia, osteogenesis imperfecta, neurofibromatosis type I, atypical osteoporotic fractures, osteoporotic fractures, atypical femoral fracture, vertebral bone fracture, distal radial fracture, facial and cranial injuries or defects, knee osteoarthritis, periodontal disease, teeth and alveolar bone defects and/or alveolar ridge defects.
The pharmaceutical composition according to any one of claims 1-15 or the therapeutic mixture according to claim 16 for use in spinal fusion surgery, maxilla-cranial reconstruction, high tibial osteotomy, periodontal repair, dental/bone implants, and/or alveolar-ridge augmentation.
The pharmaceutical composition according to any one of claims 1-15 or the therapeutic mixture according to claim 16 for use in enhancing spinal fusion rate and accelerating spinal fusion after surgeries for treating Degenerative Disc Disease.
The pharmaceutical composition or the therapeutic mixture for use according to claim 20, wherein the surgeries comprising lumbar interbody fusion (LIF) , posterolateral lumbar interbody fusion (PLIF) , anterior lumbar interbody fusion (ALIF) , oblique lumbar interbody fusion (OLIF) , extreme lateral lumbar interbody fusion (XLIF) , direct lateral interbody fusion (DLIF) , and/or open transforaminal lumbar interbody fusion (TLIF) .
Use of the pharmaceutical composition according to any one of claims 1-15 or the therapeutic mixture according to claim 16 in the manufacture of a medicament for the treatment of bone-related disorder, inducing new bone formation, promoting bone growth and/or facilitating procedures for generating or restoring bone at a particular site in an individual in need of treatment thereof.
The use according to claim 22, wherein the bone-related disorder selected from bone defects, degenerative disc disease, spinal non-union or delayed union after spinal fusion surgery, adult scoliosis, trauma (spine reconstruction) , pseudo-arthrosis associated with long bone and spine, tibial non-union fracture, hypophosphatasia, osteogenesis imperfecta, neurofibromatosis type I, atypical osteoporotic fractures, osteoporotic fractures, atypical femoral fracture, vertebral bone fracture, distal radial fracture, facial and cranial injuries or defects, knee osteoarthritis, periodontal disease, teeth and alveolar bone defects and/or alveolar ridge defects.
The use according to claim 22, wherein the treatment of the bone-related disorder selected from spinal fusion surgery, maxilla-cranial reconstruction, high tibial osteotomy, periodontal repair, dental/bone implants, and/or alveolar-ridge augmentation.
The use according to claim 22, wherein the treatment of the bone-related disorder selected from enhancing spinal fusion rate and accelerating spinal fusion after surgeries for treating Degenerative Disc Disease.
The use according to claim 25, wherein the surgeries comprising lumbar interbody fusion (LIF) , posterolateral lumbar interbody fusion (PLIF) , anterior lumbar interbody fusion (ALIF) , oblique lumbar interbody fusion (OLIF) , extreme lateral lumbar interbody fusion (XLIF) , direct lateral interbody fusion (DLIF) , and/or open transforaminal lumbar interbody fusion (TLIF) .
A method for treating bone-related disorder, inducing new bone formation, promoting bone growth and/or facilitating procedures for generating or restoring bone at a particular site in an individual in need of treatment thereof wherein the method comprising steps of:

administrating the pharmaceutical composition according to any one of claims 1-15 or the therapeutic mixture according to claim 16 to the particular site in an individual in need of treatment for the bone-related disorder.
The method according to claim 27, wherein the method further comprising the step of:

implanting an autologous bone, an allograft bone, a xenograft bone, an artificial bone, or combinations thereof.
The method according to claim 27 or 28, wherein the method further comprising the step of:

implanting an interbody fusion device.
The method according to any one of claims 27-29, wherein the particular site is the cleared intervertebral space.
The method according to any one of claims 27-30, wherein the bone-related disorder selected from bone defects, degenerative disc disease, spinal non-union or delayed union after spinal fusion surgery, adult scoliosis, trauma (spine reconstruction) , pseudo-arthrosis associated with long bone and spine, tibial non-union fracture, hypophosphatasia, osteogenesis imperfecta, neurofibromatosis type I, atypical osteoporotic fractures, osteoporotic fractures, atypical femoral fracture, vertebral bone fracture, distal radial fracture, facial and cranial injuries or defects, knee osteoarthritis, periodontal disease, teeth and alveolar bone defects and/or alveolar ridge defects.
The method according to any one of claims 27-30, wherein the treatment of the bone-related disorder selected from spinal fusion surgery, maxilla-cranial reconstruction, high tibial osteotomy, periodontal repair, dental/bone implants, and/or alveolar-ridge augmentation.
The method according to any one of claims 27-30, wherein the treatment of the bone-related disorder selected from enhancing spinal fusion rate and accelerating spinal fusion after surgeries for treating Degenerative Disc Disease.
The method according to claim 33, wherein the surgeries comprising lumbar interbody fusion (LIF) , posterolateral lumbar interbody fusion (PLIF) , anterior lumbar interbody fusion (ALIF) , oblique lumbar interbody fusion (OLIF) , extreme lateral lumbar interbody fusion (XLIF) , direct lateral interbody fusion (DLIF) , and/or open transforaminal lumbar interbody fusion (TLIF) .