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WO2021148779A1 - Système d'administration de polyhédrine libérant des facteurs de croissance - Google Patents

Système d'administration de polyhédrine libérant des facteurs de croissance Download PDF

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Publication number
WO2021148779A1
WO2021148779A1 PCT/GB2021/050112 GB2021050112W WO2021148779A1 WO 2021148779 A1 WO2021148779 A1 WO 2021148779A1 GB 2021050112 W GB2021050112 W GB 2021050112W WO 2021148779 A1 WO2021148779 A1 WO 2021148779A1
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bmp
cartilage
gdf
composition
pbmp
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Inventor
Michael Jones
Christian Pernstich
Ciara WHITTY
Raj Gandhi
Frances HENSON
Andrew MCCASKIE
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CELL GUIDANCE SYSTEMS Ltd
Cambridge Enterprise Ltd
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CELL GUIDANCE SYSTEMS Ltd
Cambridge Enterprise Ltd
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Priority to EP21702301.9A priority Critical patent/EP4093425A1/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1875—Bone morphogenic factor; Osteogenins; Osteogenic factor; Bone-inducing factor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein

Definitions

  • the present invention relates to compositions and methods for the treatment of cartilage diseases or injuries.
  • the present invention relates to treatment of cartilage defects and osteoarthritis in a subject.
  • the invention can be used in research, medical applications and veterinary applications.
  • OA Osteoarthritis
  • cartilage degradation typified by cartilage degradation, synovial inflammation and changes to subchondral bone. It is a progressive degenerative disease and approximately 8.75 million people in the UK aged 45 years and over have sought treatment for OA, with symptoms of joint stiffness, impaired mobility and persistent pain, all of which contribute to a diminished work capacity and quality of life(1 ).
  • Standard treatments include pain management, anti-inflammatory medication, lifestyle changes and regenerative medicine strategies. As the symptoms increase and non-surgical therapies are no longer effective, joint replacement surgery is the standard intervention.
  • OA can be caused by joint surface defects and one experimental approach to treat these in recent years has been to use cellular scaffold implants to promote cartilage and bone growth.
  • These scaffolds can be made from a range of materials, including ex vivo collagen preparations, hydrogels, polymers and ceramics.
  • Bone morphogenetic proteins are a class of growth factor which are known to upregulate chondrocyte proliferation, stimulate cartilage growth, promote recruitment of chondroprogenitors, and up-regulate synthesis of extracellular matrix (ECM) components including collagen fibres and proteoglycans. They have also been known to exhibit chondroprotective activity in vivo (2-6).
  • ECM extracellular matrix
  • One such scaffold is disclosed in WO 2012/096997 which is an implantable metal/polymer scaffold for synovial joint repair.
  • WO2018/215752 discloses implantable hydrogel scaffolds for tissue repair such as bone or cartilage in conditions such as osteoarthritis.
  • US8916228B2 discloses biomedical scaffolds with porous layers for use in treating tissue defects.
  • the microchannels allow ingress and habitation of cells and growth factors to promote bone regeneration in the area surrounding the scaffold.
  • US6911212B2 discloses a malleable bone putty designed to be retained at the site of repair and not be washed away by blood or fluids brought to the site by the healing mechanism.
  • the putty can be impregnated with materials such as TGF-beta, PDGF, BMPs, IGF-1 , antibiotics and living cells.
  • US20180256507A1 discloses corticosteroid loaded microparticles for reducing inflammation or pain in a joint.
  • the core shell structure of the microparticles allows for sustained highly localised release of the drug.
  • the present invention seeks to address some of these problems.
  • composition comprising a polyhedrin protein in combination with at least one growth factor.
  • the polyhedrin protein complex forms a crystal scaffold.
  • the growth factors can be incorporated within the polyhedrin complex.
  • the growth factor may be selected from OP-2, OP-3, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9, BMP-10, BMP-11 , BMP-12, BMP-13, BMP-15, BMP- 16, BMP-17.
  • the composition may comprise a polyhedrin protein together with one or more bone morphogenic protein (BMP).
  • BMP bone morphogenic protein
  • the composition may additionally comprise any other therapeutic agents suitable for the disease to be treated and that can be formulated in the polyhedrin protein complex.
  • the composition may comprise a polyhedrin protein and BMP-7.
  • the composition may comprise polyhedrin protein and BMP-2.
  • the composition may comprise polyhedrin protein, BMP-7 and BMP-2.
  • composition comprising a polyhedrin protein in combination with at least one growth factor in the treatment of musculoskeletal conditions, disorders, diseases or injury in a subject.
  • the musculoskeletal condition can be any condition sustained by a mammal such as an injury of the cartilage,
  • the composition can be used to treat any diseases where treatment requires the use of growth factors.
  • diseases such as osteoarthritis, where cartilage is damaged.
  • the treatment may involve promoting sustained cellular proliferation and/or production of extracellular matrix at the site of administration.
  • growth factors used in the compositions of the invention once delivered to the site of disease enhance the cellular proliferation and production of the extracellular matrix in the region of osteoarthritis.
  • the compositions or the invention enable an increased production of ECM components such as GAGs, hyaluronic acid, fibronectin, or chondroitin sulphate, and, upregulation in the expression of ECM genes such as COLIA1 , COL2A1 and ACAN mRNA in chondrocytes, promoting chondrogenesis, ECM synthesis, cell proliferation and healing of chondral defects at the site of administration.
  • the subject to be treated may be selected from any mammal such as humans, horses, dogs, cats and livestock, amongst others where treatment with growth factors is required.
  • a pharmaceutical formulation comprising a composition with a polyhedrin protein in combination with at least one growth factor and an evaluated pharmaceutically acceptable carrier selected.
  • the pharmaceutically acceptable carrier can be selected from a physiological solution comprising any one or a combination of glucose, dextrose, normal saline, phosphate buffered saline (PBS) or Ringer's solution, or any other suitable carrier.
  • the polyhedrin forms a crystalline scaffold complex with the growth factors comprised within the scaffolds in the formulation.
  • the pharmaceutical formulation will be optimised from the candidate formulations, namely, in a gel, hydrogel, tablet, capsule, liquid, injectable solution, suspension or powder.
  • a delivery system comprising a polyhedrin protein in combination with at least one growth factor where the growth factor is released from the polyhedrin at the site of administration in a sustained manner, in a delayed manner or in a gradient-like manner.
  • the composition is preferably delivered at the site of musculoskeletal injury, or at the site of a disease such as in the cases of cartilage defect or damage.
  • the composition and/or formulation would be delivered via intra-articular injection as directed by the results of in-vivo studies.
  • the present invention can be used to treat cartilage defects and osteoarthritis.
  • the treatment may involve promoting sustained cellular proliferation at the site of administration, of, for example, chondrocytes, chondroprogenitors, mesenchymal stem or stromal cells, and synovial cells.
  • the treatment may involve one or more of increased glycosaminoglycan (GAG) production, upregulation in the expression of ECM component genes such as COL2A1 and ACAN in chondrocytes, promoting chondrogenesis, ECM synthesis, and chondrocyte proliferation to repair chondral defects directly at or surrounding the site of administration. This occurs due to the release of bioactive PODS ® -incorporated growth factor into the surrounding environment, leading to the activation of signalling pathways to promote cartilage formation or repair.
  • GAG glycosaminoglycan
  • Bone morphogenetic proteins such as BMP-2 and BMP-7
  • BMP-2 and BMP-7 have been implicated in cartilage homeostasis and repair, and are promising OA disease modifying candidates.
  • BMPs stimulate an anabolic response in cartilage explants and articular chondrocytes, promote recruitment of chondroprogenitors, and up-regulate synthesis of ECM components including collagen fibres and proteoglycans.
  • BMP-2 and BMP-7 have been shown to be chondroprotective in small and large animal models of OA (2-6).
  • the Polyhedrin Delivery System (PODS®) (Cell Guidance Systems Ltd, UK) is a protein manufacturing platform technology which has been developed to overcome limitations of the known scaffolds and delivery systems. Viral polyhedar complexes and their methods of use are disclosed in WO 2008/105672A1 , WO 2004/063371 A1 and WO 2002/36785A1 .
  • PODS® technology harnesses polyhedrin, a component of the Bombyx mori cypovirus infectious lifecycle, where the synthesised polyhedrin protein produces a complex, highly organised crystal scaffold in which virions are constrained to protect them following release to the external environment (9).
  • This survival mechanism allows for a longer window of opportunity for the embedded virions to be ingested. Once ingested, the virions can reach target intestinal cells intact, where cargo is released enabling viral transmission.
  • This mechanism has been adapted to create PODS® proteins, which are synthesised within insect cells by co-expression of polyhedrin and a cargo protein which is incorporated via an immobilization tag which binds the polyhedrin protein (10).
  • Constraint of a growth factor within a crystalline form allows for sustained release of functional, biologically active growth factor.
  • Degradation of PODS® is mediated by cell and protease dependent mechanisms, enabling release of cargo protein at physiologically relevant levels over a period of weeks to months, from a single application of crystals as demonstrated in WO 2018/189501 , where a growth factor gradient was provided by the PODS®system.
  • the inventors have shown, for the first time, that the polyhedrin system comprising BMP-2 and BMP-7 promotes cartilage repair and can be used to treat diseases such as OA.
  • the inventors have also found that even low levels of PODS®crystals containing growth factors such as BMPs exert an enhanced, prolonged therapeutic effect on chondrocyte proliferation, ECM synthesis and cartilage repair in vitro and in vivo.
  • low levels of PODS®crystals resulted from the periodic removal of cellular media, thereby removing half of the growth factor released from the PODS®each time, yet a significant effect on ECM production was still observed.
  • the present invention is particularly useful in the treatment of cartilage-associated disorders such as osteoarthritis.
  • the methods and compositions are used for the treatment of humans or for veterinary use, such as for the treatment of animals with cartilage-associated conditions, including in mammals such as horses, canines, dogs, cats and livestock.
  • cartilage disorder or “cartilage-associated disorder” as used herein refers to a medical condition that includes as a characteristic, reduced or damaged cartilage as compared to a control or normal subject.
  • a cartilage disorder may result from disease or injury and/or reduced cartilage.
  • the cartilage-associated disorder may be osteoarthritis.
  • pharmaceutically acceptable carrier refers to the acceptance or use of the carrier in the pharmaceutical industry. Preferably the carrier is approved for use in humans.
  • exemplary carriers include physiological solutions including but not limited to glucose, dextrose, normal saline, phosphate buffered saline (PBS) or Ringer's solution.
  • therapeutically effective amount refers to an amount of an active ingredient that produces the intended result.
  • crystalline formulation refers to an ordered crystalline protein lattice consisting of polyhedrin protein.
  • Crystalline formulation in combination with hydrogel, aqueous solution, paste, liquid, other biomaterials such as electrospun fabrics may be prepared with any of the following: OP-2, OP-3, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9, BMP-10, BMP-11 , BMP-12, BMP-13, BMP-15, BMP-16, BMP-17.
  • Dosage regimens may consist of either multiple injections over a long period of time (e.g., administration once per month), or a single injection.
  • Dosage of PODS® would vary depending on the species (due to differences in joint cavity volume and size), and could vary between 0.01 ng/kg to 500 mg/kg, depending on the PODS® growth factor of interest. Each integer within that range is exemplified as preferred embodiments.
  • Figure 1 shows graphs illustrating the effect of PODS® BMP-2 (pBMP-2) on real-time primary chondrocyte proliferation.
  • Cell proliferation was monitored in real time using the xCELLigence E-plate, which converts cell impedance to cell index.
  • Chondrocytes were cultured up to 14 days with a range of pBMP-2 (25-200 ng/ml equivalent), alongside PODS® Empty (pEmpty) (200 ng/ml equivalent), and recombinant BMP-2 (100 ng/ml). No media changes were performed over the entire culture period.
  • Cell index is normalised to the initial cell seeding peak at 4 hours in Figure 1 A and Day 4 in Figure 1 B, respectively. Traces represent the mean cell index for each treatment group.
  • Figure 2 shows a graph illustrating the effect of PODS® BMP-7 (pBMP-7) on real-time primary chondrocyte proliferation.
  • Cell proliferation was monitored in real- time using the xCELLigence E-plate, which converts cell impedance to cell index.
  • Chondrocytes were cultured up to 14 days with a range of pBMP-7 (25-200 ng/ml equivalent), alongside pEmpty (200 ng/ml equivalent), and recombinant BMP-7 (200 ng/ml). No media changes were performed over the entire culture period.
  • Cell index is normalised to the initial cell seeding peak at four hours in Figure 2A and Day 4 in Figure 2B. Traces represent the mean cell index for each treatment group.
  • Figure 3 shows photographs with Alcian blue staining of chondrocytes. Alcian blue staining was performed to measure GAG content after primary chondrocytes were cultured in micromass over 21 days.
  • Figure 3A shows representative staining after culturing with pBMP-2 or pBMP-7 (both 50 ng/ml equivalent), alongside pEmpty (50 ng/ml equivalent), standard rBMP-2 (100 ng/ml) or standard rBMP-7 (200 ng/ml).
  • Figure 3B shows representative staining after culturing with a dose range of pBMP-2 or pBMP-7 (25-150 ng/ml equivalent). A half media change was performed every 3-4 days during the culture period, where standard recombinant growth factor was replenished while no additional crystals were added to PODS® treated wells.
  • Figure 4 shows the influence of PODS® growth factors on ECM mRNA expression. qRT-PCR was performed to measure ECM marker expression in chondrocytes cultured in micromass over 7 days.
  • Figure 4A shows the relative expression when cells were cultured with pBMP-2 or pBMP-7 (both 50 ng/ml equivalent), alongside pEmpty (50 ng/ml equivalent), standard rBMP-2 (100 ng/ml) or standard rBMP-7 (200 ng/ml).
  • Figures 4B and 4C illustrates the relative expression of ECM markers when chondrocytes were cultured with a dose range of pBMP-2 or pBMP-7 (25-100 ng/ml equivalent), respectively. A half media change was performed on Day 3 of the culture period, where standard recombinant growth factor was replenished while no additional PODS® crystals were added to PODS® treated wells. Points and error bars represent the mean and standard deviation.
  • mice sacrificed at eight weeks post-surgery were treated with 18.75 ng equivalent pBMP-2, pBMP-7 or pEmpty in 0.5% hydrogel. Joints were fixed, sectioned and stained with Safranin O for histological analysis of cartilage repair. Images show representative repair of cartilage for each treatment group at 4 or 8 weeks post-surgery.
  • mice sacrificed at eight weeks post-surgery were treated with pBMP-2, pBMP-7 or pEmpty in 0.5% hydrogel. 6.75x105 PODS® crystals were administered per treatment group, equivalent to 18.75 ng of protein. Joints were fixed, sectioned and stained with Safranin O for histological analysis of cartilage repair at 4 and 8 weeks. Sections were scored by two observers blinded to the group assignment to assess the level of cartilage repair and osteoarthritis. Bars show the mean score with error bars representing the standard error and ** indicates significant (p ⁇ 0.05) improvement in histological score compared with Week 4 pooled data.
  • Example 1 Synthesis of PODS® BMP-2 and PODS® BMP-7 pBMP-2 and pBMP-7 were synthesised as previously described(11) using standard methods. Both constructs contained full-length BMP-2 and BMP-7 protein (NCBI accession numbers P12643 and P18075, respectively) and were fused to the H1 incorporation tag as described in US8554493B1 .
  • Transfer DNA was co-transfected into Spodoptera frugiperda 9 (Sf9) cells with linearised baculovirus (BV) DNA using TranslT®-lnsect (Mirus Bio). Replication- competent BV was rescued by recombination between the transfer vector and linearised viral DNA. Virus was harvested and plaque purification performed to isolate a single recombinant BV. Plaques were screened and BV was harvested to infect Sf9 cells to produce PODS® crystals. Subsequently, crystals were harvested and purified by lysing Sf9 cells using successive rounds of sonication and PBS washes. Purified PODS® were sterility tested and lyophilised prior to use in experiments.
  • BV baculovirus
  • Example 2 Primary chondrocyte isolation
  • Digestion buffer consisted of Dulbecco’s Modified Eagle medium (DMEM) (1 g/l glucose) with GlutaMAXTM, pyruvate and Phenol Red (ThermoFisher Scientific) supplemented with 10% foetal bovine serum (FBS) (First Link UK), 1x penicillin/streptomycin (P/S) (ThermoFisher Scientific), and 6 mg/ml collagenase A (Sigma Aldrich). After digestion, the cell suspension was filtered through a 70 pm MACS SmartStrainer (Miltenyi Biotec), centrifuged at 300xg and washed with PBS. Isolated primary chondrocytes were resuspended and expanded in growth media (DMEM (1 g/l glucose) with GlutaMAXTM, pyruvate and phenol red supplemented with 10% FBS and 1xP/S).
  • DMEM Modified Eagle medium
  • FBS foetal bovine serum
  • P/S penicillin/s
  • Chondrocytes were cultured up to passage 3 in hypoxia (3% 02) using a HeraCellTM Vios Tri-Gas Incubator (Fisher Scientific).
  • NHAC-kn Normal Human Articular Chondrocytes isolated from a six- year old female (lot number 6F4018, Lonza) were expanded in monolayer in DMEM (1 g/l glucose) with GlutaMAXTM, pyruvate and phenol red (ThermoFisher Scientific) supplemented with 10% foetal bovine serum (FBS) (First Link UK) and 1x penicillin- streptomycin-glutamine (PSG). Following the manufacturer’s guidelines, NHAC-kn cells were cultured up to passage 15. Phagocytosis of PODS® crystals were observed within 48 h of addition.
  • Cells were treated with standard rBMP-2 (100 ng/ml) (Cell Guidance Systems), standard rBMP-7 (200 ng/ml) (ThermoFisher Scientific), PODS® Empty (pEmpty) (50 ng/ml) (Cell Guidance Systems), pBMP-2 (25-200 ng/ml) (Cell Guidance Systems), or pBMP-7 (25-200 ng/ml) (Cell Guidance Systems).
  • a half media change was performed every 3-4 days for PODS® containing wells (with no addition or replacement of crystals), or a half media change with replacement of growth factor for wells containing standard rBMP-2 or rBMP-7.
  • RNA concentration and quality was quantified using UV-spectrometry with the Nanodrop ND-2000 Spectrophotometer (ThermoFisher Scientific) and stored at -80°C. Reverse transcription was performed using the QuantiTect Reverse Transcription Kit (Qiagen) according to the manufacturer’s instructions and cDNA stored at -20°C.
  • qRT-PCR was performed using 2x Fast SYBR Green (ThermoFisher Scientific) with commercial primers for the following human genes: collagen typel (COL1A1), aggrecan (ACAN), and hypoxanthine-guanine phosphoribosyltransferase (HPRT) and b-actin (BACT) (housekeeping genes) (all obtained from ThermoFisher Scientific).
  • the collagen type II (COL2) forward and reverse primers sequences were 5'- TG G GTGTTCT ATTT ATTT ATTGTCTTCCT -3 ' (SEQ ID NO: 1 ) and 5'- GCGTTGGACTCACACCAGTTAGT-3' (SEQ ID NO: 2) respectively (Integrated DNA Technologies).
  • PCR conditions were 95°C for 20 seconds, followed by 40 cycles of 95°C for 3 seconds and 60°C for 30 seconds.
  • Alcian blue staining was assessed after 21 days of micromass culture. Cells were washed with PBS and fixed in 10% formalin for twenty minutes. After fixing, cells were washed with PBS before the addition of 1% Alcian blue (Sigma) diluted in 0.1 M HCI. This was incubated overnight at room temperature, after which cells were washed three times with 0.1 M HCI, then washed with distilled water. Alcian blue staining was visualised by bright microscopy.
  • xCELLigence® RTCA DP instrument (ACEA Biosciences Inc.). Primary OA chondrocytes or NHAC- kn cells were serum starved overnight in DMEM + 1% FBS before treatment with standard rBMP-2 (100 ng/ml), standard rBMP-7 (200 ng/ml), pEmpty (200 ng/ml equivalent, pBMP-2 (25-200 ng/ml), or pBMP-7 (25-200 ng/ml). The xCELLigence E- plate wells (ACEA Biosciences Inc.) were filled with 50 pi of DMEM + 1% FBS and incubated at room temperature for 2 hours. Cells were then seeded at 1 x 103 cells per well and incubated at room temperature for 30 minutes. Proliferation was monitored for up to 14 days without any media changes or further supplements.
  • Example 8 Histology Histological analysis was performed at Week 4 to assess the response of the joint to the administration of intra-articular PODS® and at Week 8 post-surgery to assess the rate of joint healing and OA using industry-standard scoring methods.
  • Animals were humanely sacrificed at four and eight weeks post-surgery and stifles retrieved. Knee joints were fixed and decalcified in 10% EDTA pH 8 for 14 days. The joints were processed through a series of sequential ethanol and xylene immersions with the Leica TP1020 Semi-enclosed Benchtop Tissue Processor (Leica Biosystems) and paraffin embedded using the HistoCore Arcadia (Leica Biosystems). Joints were serially sectioned at 7 pm intervals using the Leica Biosystems RM2245 Semi- Automated Rotary Microtome (Leica Biosystems). Three sections per animal were scored from the middle of the defect.
  • Sections were stained with Safranin O. After deparaffinisation and rehydration, sections were stained with Weigert’s iron haematoxylin (Sigma Aldrich) working solution for 10 minutes, followed by washing under tap water for 10 minutes. Slides were transferred to 0.1% Fast Green FCF (Sigma Aldrich) for 5 minutes, before being transferred to 1% acetic acid for 10 to 15 seconds. Subsequently slides were stained in 0.1% Safranin O solution for 5 minutes. Sections were then dehydrated through 100% ethanol, cleared with xylene and mounted using DPX mounting medium. Slides were analysed with a Nikon Eclipse Ti inverted Microscope, and images captured with an Orca OSG camera (Nikon, Japan) using NIS-Elements Advanced Research software.
  • Example 9 PODS® BMP-2 and PODS® BMP-7 stimulate chondrocyte proliferation.
  • the xCELLigence assay was used to monitor real-time changes in cellular proliferation in the presence of pBMP-2 ( Figures 1 and 2) or pBMP-7 ( Figures 3 and 4) compared to their respective standard recombinant counterparts.
  • This instrument measured changes in cell impedance (caused by changes in the total cell surface area in contact with the bottom of the well) to generate a cell index, from which cell number is inferred.
  • Chondrocytes were cultured with between 9x105-7.2x106 pBMP-2 or pBMP-7 (equivalent to 25-200 ng/ml standard recombinant growth factor), to assess whether there is a dose-dependent effect of PODS® on proliferation.
  • Chondrocytes were cultured with pBMP-2, pBMP-7, pEmpty (all 50 ng/ml equivalent), standard rBMP-2 (100 ng/ml), or standard rBMP-7 (200 ng/ml).
  • GAG production was assessed after 21 days of micromass culture using Alcian blue staining ( Figure 3A).
  • Production of collagen type I (COL1 ), collagen type II (COL2) and aggrecan (ACAN) was assessed after 7 days of micromass culture by qRT-PCR ( Figure 4A). A half media change was performed every 3-4 days, where only standard recombinant growth factor was replenished but no extra PODS® were added.
  • COL1A1 , COL2A1 and ACAN mRNA expression was up-regulated in chondrocytes cultured with either standard recombinant or PODS® formulations of BMP-2 and BMP-7, when normalising to cells cultured in growth media only ( Figure 4A).
  • COL1 A1 mRNA expression was up-regulated by a similar amount with standard recombinant and pBMP-2 (9-fold and 10-fold, respectively).
  • COL1A1 was more strongly up-regulated by standard rBMP-7 compared with pBMP-7 (110-fold and 17-fold, respectively).
  • ACAN mRNA expression increased by 1 .5-2-fold with 50 ng/ml and 75 ng/ml of pBMP- 2, relative to 25 ng/ml pBMP-2, with no change for 100 ng/ml of pBMP-2. There was no change in ACAN mRNA expression with any dose of pBMP-7. Lastly, COL2A1 mRNA increased between 1.5-3-fold, with 50, 75 and 100 ng/ml pBMP-2 treatment, and 8.5-fold and 3.5-fold with 50 or 75 ng/ml of pBMP-7, respectively.
  • C57BL/6 mice were subjected to a full-thickness cartilage defect, after which 6.75x105 PODS® crystals (18.75 ng equivalent) in PBS or 0.5% hydrogel were administered by intra-articular injection.
  • Mice were sacrificed four and eight weeks post-surgery, and joints were sectioned and stained with Safranin O (Figure 5). Sections were quantitatively assessed for evidence of inflammation and the extent of cartilage repair and osteoarthritis in the joints using the Modified Pineda score and Makin score, respectively.
  • OA is a degenerative disorder which currently lacks an effective early intervention to repair cartilage defects and halt progression of OA.
  • BMP-2 and BMP-7 are promising disease-modifying candidates to treat OA and have previously been shown to be chondroprotective.
  • the present invention demonstrates the efficacy and safety of pBMP-2 and pBMP-7 in vitro as well as in vivo to promote cartilage repair.
  • Expression of ACAN was higher with both pBMP-2 and pBMP-7 compared with standard recombinant growth factor, whereas expression of COL2A1 was higher for standard rBMP-2 and rBMP-7 compared with pBMP-2 and pBMP-7.
  • Induction of COL1A1 was similar for both pBMP-2 and rBMP-2, whereas for BMP-7, standard recombinant protein generated a much higher increase in expression compared with PODS®-incorporated protein. This is likely to be due to the replenishment of standard recombinant on Day 3 during the half media change, with no replacement of PODS®.
  • the half media change for the PODS®-containing wells would have removed half of the released growth factor from the culture system, which would take time to replace compared to the instant replenishment for standard recombinant growth factor-containing wells.
  • this experiment may provide a better comparison between treatment groups (and better predict in vivo applications) by performing the PODS® treatment without a media change and not replenishing standard recombinant growth factor.
  • BMP-2 and BMP-7 were continuously released from PODS® crystals enabling sustained cellular proliferation over a period of two weeks, after an initial lag phase of four days.
  • Cartilage metabolism is relatively slow in comparison with other tissues (17), therefore this lag phase is likely to be because degradation of PODS® is triggered by secreted proteases, which must be synthesized and secreted by cells before growth factor is released to trigger proliferation.
  • the proliferative activity of PODS® was in stark contrast to standard recombinant BMP-2 and BMP-7 counterparts, which only promoted chondrocyte proliferation for the first two days of culture.
  • growth factors were not replenished during the experiment, and due to their short half-life and fragility were quickly degraded, leading to loss of biological activity.
  • the dose-dependent effect of PODS® growth factor in this system was evident, with the highest doses of PODS® leading to the most proliferation. This emphasizes the advantages of PODS® in this culture system without a media change, as PODS® constantly release functional growth factor at physiologically relevant levels over a sustained period of time.
  • Intra-articular injection of just BMP-7 directly to the knee has been previously investigated as a treatment for OA in Phase I and Phase II clinical trials, but has proved to be unsuccessful (NCT00456157 - Intra-articular injection of OP-1 to affected knee (1 .0 ml) using ultrasound or fluoroscopy guidance in an outpatient setting (dose escalation study), NCT01111045 - Single intra-articular knee injection of OP-1 , and NCT01133613 - single intra-articular knee injection of OP-1).
  • the Phase I trial of BMP-7 showed a trend towards a symptom response for knee OA with a lack of dose- limiting toxicity (18). However, the results from the Phase II studies were not published and no further studies have been announced.
  • BMP-7 was also initially approved by the FDA under the Humanitarian Device Exemption programme as OP- 1 Putty (or Osigraft), a product for bone fusion during spinal surgery. However, the product was later withdrawn.
  • BMP-2 applied to an ACS carrier is approved by the Food and Drug Administration as InfuseTM Bone Graft, and by the European Medicines Agency as InductOsTM. This product is indicated for certain spinal fusion procedures, sinus augmentations, alveolar ridge augmentations, and for treating acute, stabilised open tibial shaft fractures.
  • InductOsTM European Medicines Agency
  • the present invention provides a convenient, off-the-shelf outpatient therapy for cartilage defects and early OA, addressing a currently unmet medical need and reducing the healthcare burden. Furthermore, the sustained release effect of the formulation according to the present invention provides therapeutic efficacy at much lower doses, preventing adverse side effects and improving cost-effectiveness.

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  • Animal Behavior & Ethology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • General Health & Medical Sciences (AREA)
  • Orthopedic Medicine & Surgery (AREA)
  • Epidemiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
  • Rheumatology (AREA)
  • Physical Education & Sports Medicine (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicinal Preparation (AREA)

Abstract

L'invention concerne une composition comprenant un complexe protéique avec des facteurs de croissance qui sont particulièrement utiles pour traiter des maladies musculo-squelettiques et des lésions cartilagineuses, en particulier l'arthrose. L'invention concerne également un système d'administration permettant une libération prolongée des facteurs de croissance.
PCT/GB2021/050112 2020-01-21 2021-01-18 Système d'administration de polyhédrine libérant des facteurs de croissance Ceased WO2021148779A1 (fr)

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GBGB2000840.5A GB202000840D0 (en) 2020-01-21 2020-01-21 Compositions and methods

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