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WO2011113100A1 - Méthode de traitement d'un trouble osseux - Google Patents

Méthode de traitement d'un trouble osseux Download PDF

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Publication number
WO2011113100A1
WO2011113100A1 PCT/AU2011/000296 AU2011000296W WO2011113100A1 WO 2011113100 A1 WO2011113100 A1 WO 2011113100A1 AU 2011000296 W AU2011000296 W AU 2011000296W WO 2011113100 A1 WO2011113100 A1 WO 2011113100A1
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WIPO (PCT)
Prior art keywords
bone
growth factor
cells
angiogenin
human
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PCT/AU2011/000296
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English (en)
Inventor
Andrew Brown
Michelle Rowney
Matthew James Samuel Constable
Geoff Nicholson
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BARWON HEALTH
Murray Goulburn Co Opeartive Co Ltd
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BARWON HEALTH
Murray Goulburn Co Opeartive Co Ltd
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Priority claimed from AU2010901117A external-priority patent/AU2010901117A0/en
Application filed by BARWON HEALTH, Murray Goulburn Co Opeartive Co Ltd filed Critical BARWON HEALTH
Publication of WO2011113100A1 publication Critical patent/WO2011113100A1/fr
Anticipated expiration legal-status Critical
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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
    • 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
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease

Definitions

  • the present invention relates to treating bone disorders using an osteogenic agent, to implants comprising said agent and to fractions of a basic growth factor extract having osteogenic activity.
  • Bone is dynamic, undergoing a continual process of remodelling where bone is removed by the osteoclast (OC), through a process called resorption, and replaced by the osteoblast (OB) through the synthesis and mineralisation of new bone in a process called osteogenesis.
  • osteogenesis and bone resorption are balanced. This balance may be disrupted by diseases such as osteoporosis, osteopenia, Paget's disease, osteolytic metastasis in cancer patients, osteodistrophy in liver disease and the altered bone metabolism caused by renal failure or haemodialysis, bone fracture, bone surgery, aging, pregnancy, and malnutrition. Bone loss is accelerated with aging. The major bone disease in the older population is osteoporosis. This disease is characterized by extensive bone loss leading to an increase in bone fragility and a greater risk of fractures. It causes considerable pain, disability, disfigurement and loss of independence, and is a cost and burden to health services. Internationally, more than 1.5 million fractures occur every year as a result of osteoporosis.
  • angiogenin was found to be the protein primarily responsible for the inhibition of resorption found in preparations of Milk Basic Protein (MBP).
  • MBP Milk Basic Protein
  • the Morita group demonstrated angiogenin has no effect on the formation of bone resorbing TRAP +ve multinucleated cells, rather exerting its effect on the functioning of mature osteoclasts with a significant inhibition of resorption. They concede the mechanism of action is still unknown, however, they identified that angiogenin prevents mature cells from forming f-actin rings after disassociation from plastic, and demonstrated a decrease in the mRNA of TRAP and cathepsin-K, critical genes in the functional activity of resorbing osteoclasts.
  • Bone implants or implantable devices are often used to augment the natural regeneration process in the event of bone defects and injuries. These implants may be employed as anchors for dental restoration, fasteners and/or prosthesis for repair of bone fractures, joint replacements and other applications. Titanium and zirconium are generally preferred materials for such medical devices because of the biocompatibility of the material and its ability to accept attachment of new bone growth; however other materials including stainless steel alloys, cobalt-chrome-molybdenum alloy, and certain plastics such as polyethylene are used. Bone growth-stimulating agents may be coated or otherwise incorporated into the implants or at the implantation site to encourage new bone growth and facilitate attachment for integration of the implant with the patient's bone.
  • a first aspect provides a method of treating a bone disorder comprising
  • An alternative first aspect provides a basic growth factor extract or an active fraction thereof for use in treating a bone disorder.
  • a second aspect provides an implant comprising a basic growth factor extract or an active fraction thereof.
  • a third aspect provides a method of promoting bone growth between a subject's bone and an implant comprising administering a basic growth factor extract or an active fraction thereof.
  • An alternative third aspect provides the use of a basic growth factor extract or an active fraction thereof to promoting bone growth between a subject's bone and an implant.
  • a fourth aspect provides a fraction of a basic growth factor extract capable of inhibiting bone resorption, stimulating bone mineralization, stimulating osteoblast proliferation, inhibiting osteoclast development, or a combination thereof.
  • the inventors have found that the basic growth factor extract described in
  • PCT/AU91/00303 to Gropep Pty. Ltd. is capable of inhibiting osteoclast formation and bone resorption and stimulating bone mineralization by osteoblasts, thus causing a net anabolic effect on bone formation.
  • Certain fractions of the basic growth factor extract were found to have the same function. The extract and its active fractions are therefore useful to treat disorders where bone growth is advantageous, for example in the treatment of bone disorders or to encourage growth between implants and a subject's bone.
  • Figure 1 shows osteoclast formation per slice from Donor 1 (A), and Donor 2 (B), and corresponding resorption data in donor 1 (C), and Donor 2 (D) for CFU-GM (4 x
  • Figure 2 shows osteoclast formation in CFU-GM treated in (A) the absence of, and (B) in the presence of basic growth factor extract and corresponding resorption assay (C) in the absence of, and (D) in the presence of basic growth factor extract.
  • Figure 3 shows the effect of fraction SW4 in a human osteoclastogenesis assay.
  • Figure 4 shows the effect of Fraction SW5 in a human osteoclastogenesis assay.
  • Figure 5 shows rat UMR 106.01 cells (2x10 4 /well) cultured in a 96 well plate with osteogenic media and increasing concentrations of Fraction SW4.
  • Cells were assessed for viability by MTT (A), Alkaline-Phosphatase (ALP) Activity was measured in standard International units (SlU) per well (B), and also expressed as relative Alk-Phos (SlU) per well (C), and mineralisation of bone (measured as CaCI 2 mg/ well) (D). Groups with different annotations are significantly different (p ⁇ 0.05, one-way ANOVA, Fisher's Multiple
  • Figure 6 shows osteoblasts (2x10 4 /well) cultured in a 96 well plate with osteogenic media and increasing concentrations of Fraction SW4. Cells were assessed for viability by MTT (not shown), Alkaline-Phosphatase (ALP) Activity was measured in standard
  • Figure 7 shows rat UMR 106.01 cells (2x10 4 /well) or Saos-2 cells (2x10 4 /well) cultured in a 96 well plate with osteogenic media and increasing concentrations of Fraction SW5.
  • Cells were assessed for viability by MTT (not shown), Alkaline-Phosphatase (ALP) Activity was measured in standard International units (SlU) per well (not shown), and also expressed as relative Alk-Phos (SlU) per well (top), and mineralisation of bone (measured as CaCI 2 mg/ well) (bottom). Groups with different annotations are significantly different (p ⁇ 0.05, one-way ANOVA, Fisher's Multiple Comparison Test).
  • 'NEG' represents control cells cultured in media in the absence of osteogenic components.
  • Figure 8 shows the effect of 60% RNAse 5 on resoption of bone by Mature Human Osteoclasts.
  • Mature CFU-GM were cultured on dentine slices for 4 days in hM-CSF and sRANKL, with increasing concentrations of 60% RNAse 5.
  • 'CONT' represents control cells cultured with hM-CSF and sRANKL only.
  • Figure 9 shows the effect of 60% RNAse 5 in human Saos-2 osteogenesis bioassays.
  • Human Saos-2 cells (2x10 4 /well) were cultured in a 96 well plate with osteogenic media and increasing concentrations of 60% RNAse 5. Mineralisation of bone was measured as CaCI2 mg/well. Groups with different annotations are significantly different (p ⁇ 0.05, one-way ANOVA, Fisher's Multiple Comparison Test).
  • 'NEG' represents control cells cultured in media in the absence of osteogenic components.
  • Figure 10 shows the effect of 70% RIPTAC on human osteoclastogenesis.
  • Figure 1 1 shows the effect of 70% RIPTAC on mature human osteoclast survival and function.
  • Figure 12 shows the effect of 70% RIPTAC in UMR osteogenesis bioassays.
  • Figure 13 shows the effect of 70% RIPTAC in a Saos-2 bioassay excluding osteogenic stimuli.
  • Figure 14 shows the effect of 70% RIPTAC in a human Saos-2 bioassay containing osteogenic stimuli.
  • Figure 15 shows the effect of 70% RIPTAC in human primary osteogenesis bioassays.
  • Figure 16 is a comparison of bovine Angiogenin-1 on human osteoclast formation and function.
  • Figure 17 shows the effect of bovine Angiogenin-1 (160609) on mature Human osteoclast survival and function.
  • Figure 18 shows the effect of bovine Angiogenin-1 (240609) on mature Human osteoclast survival and function.
  • FIG 19 shows the effect of bovine Angiogenin-1 (160609) in human Saos-2 osteogenesis bioassays.
  • Figure 20 shows the effect of bovine Angiogenin-1 (240609) in human Saos-2 osteogenesis bioassays.
  • Figure 21 shows the effect of bovine Angiogenin-1 (160609) in primary human osteogenesis bioassays.
  • Figure 22 shows the effect of bovine Angiogenin-1 (240609) in primary human osteogenesis bioassays.
  • Figure 23 shows the effect of rh Angiogenin -1 on Human Osteoclast differentiation and function.
  • Figure 24 shows the effect of rh Angiogenin -1 on Human Osteoclast survival and function.
  • Figure 25 shows the effect of rh Angiogenin -1 in human Saos-2 osteogenesis bioassays.
  • Figure 26 shows the effect of rh Angiogenin -1 in primary human osteogenesis bioassays.
  • the invention relies on a basic growth factor extract.
  • the basic growth factor extract comprises a plurality of growth factors, said factors each having a basic to approximately neutral isoelectric point.
  • the basic growth factor extract comprises a plurality of proteins.
  • the basic growth factor extract comprises a plurality of growth factors.
  • the basic growth factor extract is derived from a milk product.
  • suitable milk products include whole milk, skim milk, buttermilk, whey (such as acid or cheese/renneted whey) or a whey derivative (such as whey protein concentrate or whey protein isolate flow through), and colostrum.
  • the milk product may be obtained from any lactating animal, e.g. ruminants such as cows, sheep, buffalos, goats, and deer, non-ruminants including primates such as a human, and monogastrics such as pigs. It is preferred that skim milk or whey derived from cow's milk is used as the starting material to provide the basic growth factor extract.
  • lactating animal e.g. ruminants such as cows, sheep, buffalos, goats, and deer
  • non-ruminants including primates such as a human
  • monogastrics such as pigs.
  • skim milk or whey derived from cow's milk is used as the starting material to provide the basic growth factor extract.
  • the basic growth factor extract is prepared from a milk product by the method described in PCT/AU91/00303, namely that the milk product is subjected to cation exchange chromatography.
  • the basic growth factor extract is prepared by contacting the milk product with a cation exchange resin such that the more basic components of the milk product are absorbed on the resin, and eluting the cation exchange resin with a buffer solution comprising 2.5% NaCI or the equivalent thereof.
  • the basic growth factor extract is further fractionated to produce an angiogenin (RNAse 5) free or angiogenin depleted (reduced) extract by the method described in PCT/AU2009/000602.
  • an angiogenin reduced basic growth factor fraction can be obtained by applying the basic growth factor fraction to a cation exchange column. The run through will be depleted for angiogenin as most or all of the angiogenin will be bound to the column.
  • angiogenin (RNAse 5) depleted or free fraction, as described in PCT/AU2007/001719, namely the milk product is subjected to cation exchange chromatography.
  • the basic growth factor extract is prepared by contacting the milk product with a cation exchange resin such that the more basic components of the milk product are absorbed on the resin, and eluting the cation exchange resin with a buffer solution comprising 2.0% NaCI or the equivalent thereof.
  • the eluant is enriched for lactoperoxidase but depleted for angiogenin (RNAse 5)
  • the basic growth factor eluant may be heated to 70 to 80 degrees C for up to 5 minutes to denature some proteins present in the sample.
  • the growth factor extract eluted may be filtered to remove salt.
  • the extract may be further fractionated, for example by size exclusion, or further cation exchange chromatography, for example using a linear salt gradient.
  • the basic growth factor extract does not contain lactoferrin. In one embodiment the basic growth factor extract does not contain or is depleted in angiogenin (RNAse 5). It may contain up to 5% less, 10% less, 15% less, 20% less, 25% less, 25% less, 30% less, 35% less, 40% less, 45% less, 50% less, 55% less, 60% less, 65% less, 70% less, 75% less, 80% less, 85% less, 90% less, 95% less, 98% less or 99% less RNAse 5 than that present in the basic growth factor extract eluted from a cation exchange column using 2.5% NaCI or equivalent.
  • RNAse 5 angiogenin
  • lactoperoxidase may contain up to 5% less, 10% less, 15% less, 20% less, 25% less, 25% less, 30% less, 35% less, 40% less, 45% less, 50% less, 55% less, 60% less, 65% less, 70% less, 75% less, 80% less, 85% less, 90% less, 95% less, 98% less or 99% less lactoperoxidase than that present in the basic growth factor extract from which it is derived.
  • the basic growth factor extract does not contain IgG and/or IgA. It may contain up to 5% less, 10% less, 15% less, 20% less, 25% less, 25% less, 30% less, 35% less, 40% less, 45% less, 50% less, 55% less, 60% less, 65% less, 70% less, 75% less, 80% less, 85% less, 90% less, 95% less, 98% less or 99% less immunoglobulin than that present in the basic growth factor extract from which it is derived.
  • the basic growth factor extract comprises proteins having a molecular weight of 80 kDa or lower, 60 kDa or lower, 40 kDa or lower, 20 kDa or lower or 15 kDa or lower. In one embodiment the basic growth factor extract comprises proteins having a molecular weight of between 15 and 78 kDa or 5 to 15 kDa.
  • the basic growth factor extract or an active fraction thereof is capable of inhibiting bone resorption, stimulating bone mineralization, stimulating osteoblast proliferation, inhibiting osteoclast development, or a combination thereof.
  • Active fraction refers to a fraction of a basic growth factor extract capable of inhibiting bone resorption, stimulating bone mineralization, stimulating osteoblast proliferation, inhibiting osteoclast development, or a combination thereof.
  • any of the components of basic growth factor extract may contain any number of conservative changes in its amino acid sequence without altering its biological properties.
  • the present invention also includes the use of variants, homologues, and fragments of any one or more of the components of the basic growth factor extract.
  • the efficacy of a basic growth factor extract or active fragment thereof useful according to this invention can be evaluated both in vitro and in vivo. Briefly, the composition can be tested for its ability to promote osteoblast proliferation or inhibit osteoclasts in vitro. For in vivo studies, the composition can be injected into an animal (e.g., a mouse) and its effects on bone tissues are then accessed. Based on the results, an appropriate dosage range and administration route can be determined.
  • an animal e.g., a mouse
  • Bone disorders that may be treated by the basic growth factor extract include osteoporosis, particularly osteoporosis in the elderly or posi-menopausai women, glucocorticoid- induced osteoporosis, osteopenia, osteoarthritis; osteoporosis-related fractures, Paget's disease, osteolytic metastasis in cancer patients, osteodistrophy in liver disease and the altered bone metabolism caused by renal failure or haemodia!ysis, bone fracture, bone surgery, aging, pregnancy, and malnutrition, e.g. rickets.
  • Further uses for the basic growth factor extract include treatment of low bone mass due to chronic glucocorticoid therapy, premature gonadal failure, androgen suppression, vitamin D deficiency, secondary hyperparathyroidism, nutritional deficiencies, and anorexia nervosa.
  • treating and “treatment” as used herein refer to reduction in severity and/or frequency of symptoms, elimination of symptoms and/or underlying cause, prevention of the occurrence of symptoms (prophylaxis) and/or their underlying cause, and
  • the present method of "treating" a disorder encompasses both prevention of the disorder in a predisposed individual and treatment of the disorder in a clinically symptomatic individual.
  • Treating covers any treatment of, or prevention of a condition in a vertebrate, a mammal, particularly a human, and includes: inhibiting the condition, i.e., arresting its development; or relieving or ameliorating the effects of the condition, i.e., cause regression of the effects of the condition.
  • “Prophylaxis” or “prophylactic” or “preventative” therapy or “prevent” or “prevention” as used herein includes preventing the condition from occurring or ameliorating the subsequent progression of the condition in a subject that may be predisposed to the condition, but has not yet been diagnosed as having it.
  • the basic growth factor extract may be provided as a pharmaceutical, veterinary or neutraceutical composition or as a food.
  • a pharmaceutical composition is one which is suitable for administration to humans.
  • a veterinary composition is one that is suitable for administration to animals.
  • Said compositions may comprise one or more carriers and optionally other therapeutic agents.
  • Each carrier, diluent, adjuvant and/or excipient may be pharmaceutically "acceptable”.
  • the basic growth factor extract may be in the form of a food, food additive, food supplement, medical food, drink, drink additive or nutraceutical composition.
  • These compositions may include any edible consumer product which is able to carry protein.
  • suitable edible consumer products include confectionary products, reconstituted fruit products, snack bars, muesli bars, spreads, dips, diary products including yoghurts and cheeses, drinks including dairy and non-dairy based drinks, milk powders, sports supplements including dairy and non-dairy based sports supplements, food additives such as protein sprinkles and dietary supplement products including daily supplement tablets.
  • Suitable nutraceutical compositions useful herein may be provided in similar forms.
  • composition may further include or is co-administered with at least one other bone-enhancing agent, such as calcium, zinc, magnesium, vitamin C, vitamin D, vitamin E, vitamin K2, or a mixture thereof.
  • at least one other bone-enhancing agent such as calcium, zinc, magnesium, vitamin C, vitamin D, vitamin E, vitamin K2, or a mixture thereof.
  • the extract or its compositions may be administered orally, topically, or
  • parenteral as used herein includes intravenous, intraarterial,
  • the term "implant” refers to a medical device, including devices used to replace or supplement damaged, diseased or missing bone or tissue. Such implants may be implemented in the spine, extremities, knees, face, teeth, hips, joints, or any other region of the body known to those in the art.
  • the implant may be a spinal implant, a hip implant, a knee implant, a facial implant, a shoulder implant, an elbow implant, cranial implant, ankle implant, a wrist implant etc.
  • the term implant is also intended to extend to bone fillers and not be limited to prosthesis.
  • the basic growth factor extract is coated, absorbed or otherwise incorporated in or on the implant.
  • the implant may be porous and the basic growth factor extract may be incorporated within the implant.
  • the basic growth factor extract is administered to the implant site, either before, after or at the same time the implant is administered.
  • Example 1 Preparation of a basic growth factor extract
  • Basic growth factor extract in accordance with the present invention may be purified from skimmed milk by means of a continuous separation process (CSEP), based on the cation exchange resin SP Sepharose Big Bead (GE Healthcare).
  • CSEP continuous separation process
  • SP Sepharose Big Bead GE Healthcare
  • the proteins captured by the chromatographic purification may be subsequently ultrafiltered, microfiltered, freeze- dried and milled.
  • bovine skim milk was collected by refrigerated transport, pasteurised and stored at less than 10 ° C.
  • Skim milk was feed onto the CSEP, which consists of 30 sub-columns on a rotating platform. Each sub-column represents a packed resin bed containing 30 L of resin.
  • the CSEP operates on a counter current mechanism. Each sub-column receives fluid from a single port and periodically moves to a new port. Milk was applied at 50 CV/h until 99 CV have been applied. Milk was then rinsed from the column at 45 CV/h until 6 CV have been applied.
  • the growth factor extract was eluted from the column with 2.5% NaCI at 45 CV/h until 6 CV had been applied.
  • the column was regenerated by applying 6.5% NaCI at 45 CV/h until 6 CV had been applied.
  • the basic growth factor extract dissolved in 2.5% NaCI was pumped into a ultrafiltration (UF) plant and the retentate concentrated to approximately 2% protein.
  • the retentate was then microfiltered through a 0.8 ⁇ and then the permeate further ultrafiltrated with the continuous addition of water until the protein was greater than 95% of solids and salt was less than 5% of solids.
  • the retentate which represents concentrated and desalted basic growth factor extract, was freeze-dried.
  • the retentate was pre-frozen for a minimum of 12 h at -18 ° C in a blast freezer and freeze-dried for 36 h at 50°C.
  • the freeze-dried product was milled to powder, and sealed into 1 kg foil pouches that have been crushed to remove excess air.
  • the SW series of fractions were prepared by size-exclusion chromatography on Superose 12 or SephacrylS-100HR (GE Healthcare, product codes 17-0536-01 and 17- 1 194-01 respectively) resin.
  • the resin was loaded into a XK26/70 column (26 mmD 700 rmmL) which had a bed depth of approximately 600mm at the time of separating the basic growth factor extract.
  • the column was equilibrated with the buffer to be used during the subsequent separation, which was 20 mM sodium phosphate buffer at 1 mL/min.
  • the basic growth factor extract as prepared according to example 1 reconstituted with 20 mM sodium phosphate 50 mg/mL was applied to the column via a 5 mL syringe inserted into a Luer-M6 fitting screwed into a T-junction inserted between the pump and column.
  • the flow rate during the separation was 1 mL/min and the separation occurred at room temperature. Fractions of 10 mL were collected and then analysed by cation exchange HPLC.
  • Example 3 Fractions of basic growth factor extract prepared by further cation exchange
  • XK50/20 column (50 mmD ⁇ 200 mmL) was loaded with SP Big Bead resin (GE Healthcare) and then attached to the binary pumps of an Agilient 1 100 series HPLC.
  • the buffers used were 50 mM NaH 2 PO 4 .H 2 0 (Buffer A, pH 7.5) and 50 mM NaH 2 PO 4 .H 2 0 + 1 M NaCI (Buffer B, pH 7.5).
  • the flow was 1 mL/min.
  • the column was loaded with basic growth factor extract prepared as described in example 1 (50 mg/mL) and then rinsed with buffer A, prior to being eluted with a linear gradient changing from 0% buffer B to 100% buffer B over approximately 30 minutes.
  • CFU-GM (4 x 10 4 /slice) from 2 donors, were cultured on dentine slices for 14 days in hM-CSF and sRANKL, with increasing concentrations of a basic growth factor extract as prepared in example 1. Osteoclast formation and bone resorption data were measured and statistical analysis (ANOVA, p ⁇ 0.05) showed that samples containing basic growth factor extract contained less osteoclasts and the dentine was less degraded when compared with control samples containing hM-CSF and sRANKL only ( Figure 1 and 2).
  • Treated cells were compared to control cells cultured with hM-CSF and sRANKL only. From these results it can be concluded SW4 inhibits osteoclastogenesis between 100mg-1 mg/ml, inhibits resorption between 1 ng-1 mg/ml and inhibits the resorbtive capacity of mature osteoclasts between 10mg-1 mg/ml.
  • SW4 inhibits osteoclastogenesis at 300mg - 1 mg/ml, causes cell death in mature osteoclasts at 1 mg/ml and inhibits the resorbtive capacity of mature osteoclasts between 10mg - 100mg/ml.
  • Example 10 Process for the extraction of an RNAse 5-enriched fraction from skim milk
  • a 10 cm deep column was packed with SP Sepharose Big Beads (GE Healthcare) such that the total bed volume of the column was 29.7 litres.
  • SP Sepharose Big Beads GE Healthcare
  • To the column a flow of skimmed cow's milk was applied at a linear flow rate of 331 cm/h (34 litres of skimmed milk per litre of resin per hour) for 2 hours until the volume of skimmed milk applied was 68 times the volume of the resin packed into the column.
  • the milk remaining in the column was removed by adding 2.5 column volumes (CV) of water at a linear flow rate of 147 cm/h (15 litres of buffer per litre of resin per hour), or 0.25 CV/min, for 10 min.
  • CV column volumes
  • RNAse 5 (angiogenin)-depleted lactoperoxidase fraction was eluted from the column with 2.5 CV of a buffer containing sodium ions equivalent to 2.0% (0.34M) NaCI, at pH 6.5, by flowing the cation buffer solution at a linear flow rate of 75 cm/h (7.5 litres of cation buffer solution per litre of resin per hour), or 0.125 CV/min, for 20 min.
  • the first 0.5 litres of cation buffer solution per litre of resin was discarded to drain and the next 2.5 litres of cation buffer solution per litre of resin was collected as the angiogenin-depleted lactoperoxidase fraction (including 0.5 litres of cation buffer solution per litre of resin overlapping the application time of the next buffer, i.e. breakthrough time).
  • RNAse 5-enriched fraction was then eluted from the column with 2.5 CV of a buffer containing sodium ions equivalent to 2.5% w/v (0.43 M) NaCI, at pH 6.5, by flowing the cation buffer solution at a linear flow rate of 75 cm/h (7.5 litres of cation buffer solution per litre of resin per hour), or 0.125 CV/min, for 20 min.
  • the first 0.5 litres of cation buffer solution per litre of resin was discarded to drain and the next 2.5 litres of cation buffer solution per litre of resin was collected as the angiogenin-enriched fraction (including 0.5 litres of cation buffer solution per litre of resin overlapping the application time of the next buffer).
  • the lactoferrin fraction is eluted from the column with 2.5 CV of a buffer containing sodium ions equivalent to 8.75% w/v (1.5 M) NaCI, at pH 6.5, by flowing the cation buffer solution at a linear flow rate of 75 cm/h (7.5 litres of cation buffer solution per litre of resin per hour), or 0.125 CV/min, for 20 min.
  • the first 0.5 litres of cation buffer solution per litre of resin was discarded to drain and the next 2.5 litres of cation buffer solution per litre of resin was collected as the lactoferrin fraction.
  • the angiogenin-enriched fraction that was collected was ultrafiltrated (NMWCO 5 kDa) to concentrate and reduce the salt content.
  • the resultant concentrate was freeze-dried and stored at room temperature for subsequent use.
  • the angiogenin-enriched fraction was analysed for angiogenin content by SDS- PAGE, and the fraction was found to contain 60% (protein basis) of a low molecular weight (14 kDa) protein which was confirmed to be angiogenin (RNAse 5) by MALDI-TOF/TOF MS.
  • RNAse 5 does not alter bone resoption by osteoclasts
  • RNAse 5 Mature human osteoclasts were grown in cell culture with various concentrations of RNAse 5 ( Figure 8) prepared as described in example 10. Specifically, mature CFU-GM were cultured on dentine slices for 4 days in hM-CSF and sRANKL, with increasing concentrations of 60% RNAse 5. When cell cultures not containing RNAse 5 were compare to those containing RNAse 5 it was apparent that RNAse 5 had no significant effect on resorption due to osteoclasts (p ⁇ 0.05).
  • RNAse 5 does not alter bone mineralisation by osteoblasts
  • RNAse 5 Mature human osteoblasts were grown in cell culture with various concentrations of RNAse 5 ( Figure 9) prepared as described in example 10. Specifically, human Saos-2 cells (2x10 4 /well) were cultured in a 96 well plate with osteogenic media and increasing concentrations of 60% RNAse 5. Mineralisation of bone was measured as CaCI 2 mg/well. When cell cultures not containing RNAse 5 were compare to those containing RNAse 5 it was apparent that RNAse 5 had no significant effect on bone mineralisation due to osteoblasts (p ⁇ 0.05).
  • Example 13 Growth factor extract activity in human osteoclasts
  • RIPTAC is an angiogenin enriched bovine milk extract made according to the process described in PCT/AU2007/001719. RIPTAC was received in a powdered form reportedly containing 70% angiogenin-1 , and was initially used to determine the effects of angiogenin-1 in the absence of a pure source.
  • CFU-GM (4 x10 4 ) were cultured on dentine slices for 14 days in hM-CSF and sRANKL with increasing concentrations of 70% RIPTAC.
  • RIPTAC exhibited minimal influence on the formation of TRAP+ve multinucleated cells in culture. There was a dose dependant decrease in both mean cell size (Figure10(B)) and total OCL plan area (Figure 10(C)), with no effect on total cell number ( Figure 10(A)). There was a significant decrease in resorption by cells treated with 1 mg/ml of RIPTAC ( Figure 10(D)) only.
  • Example 14 Effect of RIPTAC on mature cell survival and ability to resorb bone
  • RIPTAC exhibited only minimal biological effect on mature human osteoclasts (Figure 1 1 ).
  • RIPTAC decreased the survival (Figure 1 1 (A)) of osteoclasts at the highest dose of 1 mg/ml, with no real effect on either mean cell size (Figure 1 1 (B)) or total OC plan area (Figure 1 1 (C)).
  • Morita et al demonstrated the inhibition of resorption in mature cells with an IC 50 of 3 ⁇ g/ml, however, in the human model we have demonstrated no significant effect of the 70% purified angiogenin over the entire range tested. Interestingly, we could discern a decreasing trend in resorption inhibition ⁇ g - 1 mg/ml.
  • Rat UMR 106.01 cells (2x10 4 /well) were cultured in a 96 well plate with osteogenic media and increasing concentrations of RIPTAC.
  • Cells were assessed for viability by MTT (A), Alkaline-Phosphatase (ALP) activity was measured in standard International units (SlU) per well (B), and also expressed as relative Alk-Phos (SlU) per well (C), and mineralisation of bone was measured as CaCI 2 [ ⁇ gl well (D). Groups with different annotations are significantly different (p ⁇ 0.05, one-way ANOVA, Fisher's Multiple Comparison Test).
  • 'NEG' represents control cells cultured in media in the absence of osteogenic components.
  • the initial screen of RIPTAC in the UMR system yielded interesting results.
  • RIPTAC had no effect on cell proliferation 1 ng - 10C ⁇ g/ml (Figure 12(A)) in comparison to the osteogenic control; however, there was a significant increase in the total viable cells in the highest treatment of 1 mg/ml which was not significant as compared to the negative control. There was, however, a significant decrease in the ALP per well in the treatments 1 ng - 10C ⁇ g/ml ( Figure 12(B)) as compared to the osteogenic control, with a significant increase in ALP at the highest dose of 1 mg/ml. There were no discernible trends in the relative ALP per well (Figure 12(C)).
  • Example 16 Effect of 70% RIPTAC in a Saos-2 bioassay excluding osteogenic stimuli
  • Example 17 Effect of 70% RIPTAC in a Saos-2 bioassay containing osteogenic stimuli
  • Human Saos-2 cells (2x10 4 /well) were cultured in a 96 well plate with osteogenic media and increasing concentrations of RIPTAC.
  • Cells were assessed for viability by MTT (A), Alkaline-Phosphatase (ALP) Activity was measured in standard International units (SIU) per well (B), and also expressed as relative Alk-Phos (SIU) per well (C), and mineralisation of bone was measured as CaCI 2 [ ⁇ gl well (D). Groups with different annotations are significantly different (p ⁇ 0.05, one-way AN OVA, Fisher's Multiple Comparison Test).
  • 'NEC represents control cells cultured in media in the absence of osteogenic components.
  • Example 18 The Effect of RIPTAC in human primary osteogenesis bioassays
  • CFU-GM (4 x 10 4 ) were cultured on dentine slices for 14 days in hM-CSF and sRANKL with increasing concentrations of bovine Angiogenin either batch 160609 or 240609.
  • the two Angiogenin-1 preparations both inhibited cell formation at the highest dose of 100 ⁇ g/ml ( Figure 16(A)), with no difference as compared to control over the remaining dose range (100ng/ml - 10 ⁇ g/ml).
  • Figure 16(B) Mean cell size
  • Figure 16(C) % TRAP
  • Figure 16(D) % Resorption
  • Angiogenin-1 is specific to the functioning of mature Osteoclasts. Accordingly it was important to determine the effects of purified bovine Angiogenin-1 batches in our mature human Osteoclast model.
  • Example 21 Effect of bovine Angiogenin-1 (240609) on mature Human Osteoclast survival and function
  • Bovine Angiogenin-1 samples were assessed for their ability to stimulate mineralisation in the Saos-2 human Osteosarcoma cell Line, and the effect of batch 160609 is reported below in Figure-19.
  • Human Saos-2 cells (2x10 4 /well) were cultured in a 96 well plate with osteogenic media and increasing concentrations of bovine Angiogenin-1 (batch 160609). Cells were assessed for viability by MTT (A), Alkaline-Phosphatase (ALP) Activity was measured in standard International units (SlU) per well (B), and also expressed as relative Alk-Phos (SlU) per well (C), and mineralisation of bone was measured as CaCI 2 ⁇ g/ well (D). Groups with different annotations are significantly different (p ⁇ 0.05, one-way ANOVA, Fisher's Multiple Comparison Test). 'NEC represents control cells cultured in media in the absence of osteogenic components.
  • Example 24 Effect of bovine Angiogenin-1 (Batch 160609) in primary human
  • BC4 Primary human Osteoblast cells (2x10 4 /well) were cultured in a 96 well plate with osteogenic media and increasing concentrations of bovine Angiogenin-1 (batch 160609). Cells were assessed for viability by MTT (A), Alkaline-Phosphatase (ALP) Activity was measured in standard International units (SlU) per well (B), and also expressed as relative Alk-Phos (SlU) per well (C), and mineralisation of bone was measured as CaCI 2 ⁇ g/ well (D). Groups with different annotations are significantly different (p ⁇ 0.05, one-way ANOVA, Fisher's Multiple Comparison Test). 'NEC represents control cells cultured in media in the absence of osteogenic components.
  • Example 25 Effect of bovine Angiogenin-1 (Batch 240609) in primary human
  • CFU-GM were cultured on dentine slices for 14 days in hM-CSF and sRANKL, with increasing concentrations of recombinant human Angiogenin-1 .
  • Example 27 The effect of rhAngiogenin-1 on mature Human Osteoclast survival and
  • Example 28 The effect of human Angiogenin-1 in Saos-2 osteogenesis bioassays
  • Example 29 The effect of human recombinant Angiogenin-1 in primary human

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Abstract

La présente invention concerne une méthode de traitement d'un trouble osseux qui comprend l'administration, à un sujet en ayant besoin, d'une quantité efficace d'un extrait de facteur de croissance de base ou de sa fraction active. L'invention concerne en outre un implant qui comprend un extrait de facteur de croissance de base ou sa fraction active.
PCT/AU2011/000296 2010-03-17 2011-03-17 Méthode de traitement d'un trouble osseux Ceased WO2011113100A1 (fr)

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WO2008055310A1 (fr) * 2006-11-10 2008-05-15 Murray Goulburn Co-Operative Co. Limited Procédé de préparation de l'angiogénine
WO2009137879A1 (fr) * 2008-05-14 2009-11-19 Agriculture Victoria Services Pty Ltd Formes posologiques administrables par voie orale comprenant de l'angiogénine et utilisations correspondantes

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WO2008055310A1 (fr) * 2006-11-10 2008-05-15 Murray Goulburn Co-Operative Co. Limited Procédé de préparation de l'angiogénine
WO2009137879A1 (fr) * 2008-05-14 2009-11-19 Agriculture Victoria Services Pty Ltd Formes posologiques administrables par voie orale comprenant de l'angiogénine et utilisations correspondantes

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2016500662A (ja) * 2012-10-08 2016-01-14 マレー・ゴールバーン・コー−オペラティヴ・カンパニー・リミテッド 乳汁から成長因子を精製するための改良されたプロセスおよびその製品

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