Hibiscus Extract for Use in Bone and Cartilage Repair

(19) TZZ Z_ _T

(11) EP 2 509 612 B1

(12) EUROPEAN PATENT SPECIFICATION

(45) Date of publication and mention (51) Int Cl.: of the grant of the patent: A61K 36/185 (2006.01) A61K 36/889 (2006.01) 21.11.2018 Bulletin 2018/47 A61K 36/48 (2006.01) A61P 19/04 (2006.01) A61P 19/08 (2006.01) (21) Application number: 10835314.5 (86) International application number: (22) Date of filing: 10.12.2010 PCT/AU2010/001679

(87) International publication number: WO 2011/069214 (16.06.2011 Gazette 2011/24)

(54) HIBISCUS EXTRACT FOR USE IN BONE AND CARTILAGE REPAIR HIBISKUS EXTRAKT ZUR VERWENDUNG IN KNOCHEN- UND KNORPELREPARATUR EXTRAIT D’HIBISCUS POUR L’UTILISATION DANS LA RÉPARATION DE L’OS ET DU CARTILAGE

(84) Designated Contracting States: • WHISTLER ET AL: "Traditional and herbal AL AT BE BG CH CY CZ DE DK EE ES FI FR GB medicine in the cook islands", JOURNAL OF GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO ETHNOPHARMACOLOGY, ELSEVIER PL PT RO RS SE SI SK SM TR SCIENTIFIC PUBLISHERS LTD, IE, vol. 13, no. 3, 1 July 1985 (1985-07-01), pages 239-280, (30) Priority: 10.12.2009 AU 2009906034 XP025531157, ISSN: 0378-8741, DOI: 10.1016/0378-8741(85)90072-8 [retrieved on (43) Date of publication of application: 1985-07-01] 17.10.2012 Bulletin 2012/42 • Smith, A.C.: "Flora Vitiensis Nova: A New Flora of Fiji (Spermatophytes Only), Volume 2", 1981, (73) Proprietor: Cimtech Pty Limited Pacific Tropical Botanical Garden, Lawai, Hawai, Newcastle West, New South Wales 2302 (AU) XP002701182, vol. 2, page 417, * page 417 * • LI, L ET AL.: ’Structure elucidation of a new (72) Inventors: friedelane triterpene from the mangrove plant • MATHESON, Graham Hibiscus tiliaceus’ MAGNETIC RESONANCE IN Cronulla, CHEMISTRY vol. 44, 2006, pages 624 - 628, New South Wales 2230 (AU) XP008161288 • WALSH, William • CHEN, J ET AL.: ’A New Cytotoxic Amide from Randwick, the Stem Wood of Hibiscus tiliaceus’ New South Wales 2031 (AU) PLANTA.MED vol. 72, 2006, pages 935 - 938, XP008159081 (74) Representative: Lavoix • ROSA, RM ET AL.: ’Antioxidant and Bayerstrasse 83 Antimutagenic Properties of Hibiscus tiliaceus L. 80335 München (DE) Methanolic Extract’ J. AGRIC. FOOD CHEM vol. 54, 2006, pages 7324 - 7330 (56) References cited: • MELECCHI, MIS ET AL.: ’Optimization of the WO-A1-2010/127396 US-A1- 2009 155 216 sonication extraction method of Hibiscus tiliaceus L. flowers’ ULTRASONICS SONOCHEMISTRY vol. 13, 2006, pages 242 - 250, XP028073634

Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the Implementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 2 509 612 B1

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Description 1985) discloses that in Maori medicine chewed or mashed flowers Hibicus tiliaceus L. are applied with or Field of the Invention without coconut oil to boils, carbuncles and cuts; it is further disclosed that a solution of grated bark of Hibiscus [0001] The present invention relates generally to plant 5 tiliaceus L. combined with the crushed bark or husk of extracts and compositions comprising the same for use nù (= Cocos nucifera L.) in water is used to soak bone in the treatment of bone and cartilage injuries, diseases fractures and sprains. or defects, the promotion of healing of bone and cartilage [0007] Smith, A.C. ("Flora Vitiensis Nova: A new Flora injuries, and the promotion of bone and cartilage forma- of Fiji (Spermatophytes Only), Volume 2, 1981, Pacific tion. Particular embodiments as disclosed herein find ap- 10 Tropical Botanical Garden, Lawai, Hawaï vol. 2, page plication in, inter alia, the treatment of bone fractures and 417) describes that in medicine on Fiji the leaves of Hi- in bone formation and regeneration. biscus tiliaceus are wrapped on bone fractures. [0008] US 2009/155216 discloses the use of embry- Background of the Invention onic stem cells, mesenchymal stem cells, osteoblasts, 15 pre-osteoblasts or chondrocytes in the treatment of bone [0002] Bone is a living tissue comprising a number of diseases including bone fracture and osteoporosis. constituents including calcium carbonate, calcium phos- [0009] The present inventors have surprisingly found phate, collagen and water and is continuously being re- that plant extracts disclosed herein and compositions plenished by resorption and deposition of bone matrix. comprising same, promote bone and cartilage repair by [0003] When a bone fractures the usual treatment is 20 inducing new bone formation and new cartilage growth. to reposition the fractured bone back into place, to sta- bilise the position of the bone, and then to wait for the Summary of the Invention bone healing process to occur. Bone healing is a complex process which is generally regarded as involving three [0010] The invention is as defined in the claims. phases: reactive phase, reparative phase and remodel- 25 [0011] The extract may further comprise an extract of ling phase. one or more of Vigna marina (Burm.) Merr., Cocos nuci- [0004] During the reactive phase a haematoma forms fera L., or Terminalia catappa L. at the fracture site and inflammatory cells and fibroblasts [0012] The extract(s) may be derived from one or more infiltrate the bone under prostaglandin mediation. This of the following: bark, leaf, vine, bean, husk or nut. In one results in the formation of granulation tissue, ingrowth of 30 embodiment the Hibiscus tiliaceus L. extract is derived vascular tissue, and migration of mesenchymal cells. from bark, typically fresh crushed bark. During thereparative phase, fibroblasts begin to laydown [0013] The extract may be prepared using and/or com- stroma that helps support vascular ingrowth. As vascular prises,a plant basedoil, a hydrocarbon and/oran alcohol. ingrowth progresses, a collagen matrix is laid down while The plant based oil may be derived from plant seeds or osteoid is secreted and subsequently mineralised, which 35 fruit. In a particular embodiment the plant based oil is leads to the formation of a soft callus (cartilage) around derived from Cocos nucifera. The Cocos nucifera oil may the repair site. After a period of time from weeks to months be, for example, virgin Cocos nucifera oil, refined Cocos the cartilage ossifies, forming a bridge of woven bone nucifera oil, hydrogenated Cocos nucifera oil or fraction- between the fracture fragments. During the remodelling ated Cocos nucifera oil. phase the healing bone is restored to its original shape, 40 [0014] The composition may be formulated so as to be structure, and mechanical strength. The remodelling suitable for administration by any route, for example top- phase occurs slowly over months to years, however, ad- ical or parenteral. Parenteral administration may com- equate bone strength is typically achieved in three to six prise, for example, intraosseous infusion or intrathecal months. injection. [0005] The healingof bonefractures is clearlya lengthy 45 [0015] Disclosed herein is a method for promoting the process. Notably, cartilage, unlike other connective tis- formation, repair or regeneration of bone or cartilage in sues, does not contain blood vessels and therefore com- a subject, the method comprising administering to the pared to other connective tissues, grows and repairs subject in need thereof an effective amount of an extract, more slowly. Further, bone healing can be delayed or a composition , or osteoblasts, osteoblastic progenitor impaired when any of the healing processes do not func- 50 cells, or bone segments or fragments cultured in the pres- tion properly or in a timely manner. This can be both a ence of said extract or composition, as defined herein major setback to the individual and a costly clinical prob- [0016] The subject may be suffering from a bone or lem. Accordingly, approaches that speed up the body’s cartilage injury, defect or disease or may be susceptible natural process of regenerating bone and cartilage are or predisposed to such an injury, defect or disease. The clearly advantageous. 55 injury may be, for example, a bone fracture. The disease [0006] Whistler et al. ("Traditional and herbal medicine may be a degenerative bone disease, for example, os- in the cook islands", Journal of Ethnopharmacology, El- teoporosis. The subject may be undergoing or have un- sevier Scientific Publishers Ltd, IE, vol. 13, no. 3, 1 July dergone a bone or cartilage graft procedure, such as a

2 3 EP 2 509 612 B1 4 skeletal fusion procedure, for example a spinal fusion coconut oil. The skin samples were stained with he- procedure. matoxylin and eosin and are shown at x400 magni- [0017] The osteoblasts or osteoblastic progenitor cells fication. may be autogeneic, allogeneic or xenogeneic. [0018] Disclosed herein is a method for treating a bone 5 Figure 2 shows histologic profiles of skin of 12 week or cartilage injury, defect or disease in a subject, the old female New Zealand rabbits. A and B, no treat- method comprising administering to the subject an effec- ment; C and D, daily application for 7 days of a com- tive amount of an extract, a composition, or osteoblasts, bination extract of Vigna marina (Burm.) Merr., Co- osteoblastic progenitor cells, or bone segments or frag- cos nucifera L., Terminalia catappa L. and Hibiscus ments cultured in the presence of said extract or compo- 10 tiliaceus L. in coconut oil; E and F, daily application sition, as defined herein. for 7 days of Hibiscus tiliaceus L. in coconut oil. The [0019] The injury may be, for example, a bone fracture. skin samples were stained with hematoxylin and The disease may be a degenerative bone disease, for eosin and are shown at x40 (A, C, E) and x400 (B, example, osteoporosis. The extract or composition may D, F) magnification. enhance the rate or extent of healing of the injury or dis- 15 ease. The extract or composition may promote bone or Figure 3 shows histologic differences in untreated cartilage formation, repair or regeneration. (A and B) and treated (C to F) 18 month old oestro- [0020] Disclosed herein is a method for promoting gen deficient (ovaries removed at age 6 weeks) rats; bone or cartilage growth surrounding a bone or cartilage and untreated (G to H) 18 month old non-oestrogen graft or implantable device, the method comprising ad- 20 deficient (control surgical procedure carried out at ministering to a subject prior to, during or after a graft or age 6 weeks and rats allowed to recover) and treated implantation procedure, an effective amount of extract, rats (I to L), following a surgically created fracture of a composition , or osteoblasts, osteoblastic progenitor the femur by osteotomy, and operative repair with cells or bone segments or fragments cultured in the pres- internal fixation. The treated rats were subjected to ence of said extract or composition, as defined herein. 25 a daily application of an extract of Hibiscus tiliaceus [0021] The graft may be an autograft or allograft. The L. in coconut oil to the fracture site for 21 days. The graft procedure may be, for example, for the repair of a fracture histology samples were stained with hema- bone fracture or for the purposes of skeletal fusion such toxylin and eosin (A, C, E, G, I, K) and Saffron-O as spinal fusion. The implantable device may be a bone Stain (B, D, F, H, J, L) which stains proteoglycan in fixation device or a prosthesis. The extract according to 30 cartilage purple; and are shown at x12 magnification. the first aspect, the composition according to the second aspect, or osteoblasts, osteoblastic progenitor cells or Figure 4 shows a graph of histological scoring at the bone segments or fragments cultured in the presence of fracture sites in untreated 18 month old oestrogen said extract or composition may be coated on to the pros- deficient (ovaries removed at age 6 weeks) rats (Ovx thesis, used as an adjunct to a bone fixation device, or 35 Control) (control surgical procedure was carried out used as an adjunct to a bone cement used to anchor the at age 6 weeks and rats allowed to recover); and prosthesis or bone fixation device. treated 18 month old oestrogen deficient (ovaries [0022] Administration to the subject may be during an removed at age 6 weeks) rats (Ovx rx) rats, following arthroscopic or open surgical procedure. a surgically created fracture of the femur by osteot- [0023] Disclosed herein is a method for the formation 40 omy, and operative repair with internal fixation. The or growth of bone, the method comprising incubating os- treated rats were subjected to a daily application of teoblasts, osteoblastic progenitor cells or bone segments an extract of Hibiscus tiliaceus L. in coconut oil to or fragments in the presence of an effective amount of the fracture site for 3 weeks. an extract or a composition as defined in claim 1, under conditions suitable to induce the formation or growth of 45 Figure 5 shows computer models A( and C) and bone. radiographs (B and D) of surgically created 20mm defects of the ulna (including periosteum) by osteot- Brief Description of the Drawings omy, and with no graft or substitute filler in 12 week old NZ white rabbits, following a daily application for [0024] The present invention will now be described, by 50 7 days to the epithelial surface of the back, not on way of non-limiting example only, with reference to the the limb of the fracture site of: A and C; coconut oil accompanying drawings in which: with intermediate hydrocarbon Extract and; C to D, hydrocarbon extract of Hibiscus tiliaceus L. in coco- Figure 1 shows histologic profiles of skin of 10 to 14 nut oil. weeks old female rats. A, no treatment; B, daily ap- 55 plication for 7 days of coconut oil; C, daily application Figure 6 shows histologic profiles in 12 week old for 7 days of Hibiscus tiliaceus L. in ethanol; and D, female New Zealand rabbits following a daily appli- daily application for 7 days of Hibiscus tiliaceus L. in cation for 7 days to the epithelial surface at the frac-

3 5 EP 2 509 612 B1 6 ture site (surgically created 20mm defect of the ulna Figure 10 shows histologic profiles of the spine of (including periosteum) by osteotomy, and with no female New Zealand rabbits which underwent bilat- graft or substitute filler) of: a combination extract of eral single level postero-lateral fusion using Vigna marina (Burm.) Merr., Cocos nucifera L., Ter- autograft harvested from the iliac crest at 12 weeks minalia catappa L. and Hibiscus tiliaceus L. in coco- 5 old. Surgery was followed by routine care ( A) or daily nut oil (C and D); an extract of Hibiscus tiliaceus L. application for 5 days and then twice weekly appli- in coconut oil ( E and F). A and B, untreated controls. cation to 6 weeks to the epithelial surface rostral to, The samples were stained with pentachrome and but not overlying the fracture site of 5ml of an extract are shown at x12.5 (A, C, E) and x25 (B, D, F) mag- of Hibiscus tiliaceus L. in coconut oil following ex- nification. 10 traction in ethanol and hydrocarbon (B). The samples were stained with hematoxylin and eosin and Figure 7 shows histologic profiles in 12 week old are shown as a composite of images at x12.5 mag- female New Zealand rabbits following a daily appli- nification showing the transverse process and the cation to the epithelial surface at the fracture site spinal fusion mass 2-3 mm lateral to the vertebral (surgically created surgically created 20mm defect 15 body. of the ulna (including periosteum) by osteotomy, and with no graft or substitute filler) for 7 days of: A and Figure 11 show the results of mechanical testing of B, a combination extract ofVigna marina (Burm.) treated autografts at 8 weeks and untreated controls Merr., Cocos nucifera L., and Terminalia catappa L. at 12 weeks. (no Hibiscus tiliaceus L.) in coconut oil; C and D, an 20 extract of Hibiscus tiliaceus L. in ethanol; E and F, Figure 12 show mass chromatograms of: A, Hibis- an extract of Hibiscus tiliaceus L. in coconut oil fol- cus tiliaceus L. in coconut oil following extraction in lowing extraction in ethanol; and G and H, an extract ethanol and hydrocarbon and; B, the hydrocarbon of Hibiscus tiliaceus L. in coconut oil following ex- intermediate layer. traction in ethanol and hydrocarbon. The samples 25 were stained with pentachrome and are shown at Detailed Description of the Invention x12.5 magnification. [0025] The articles "a" and "an" are used herein to refer Figure 8 shows a graph of the bone growth score in to one or to more than one (i.e., to at least one) of the surgically created 20mm defects of the ulna (includ- 30 grammatical object of the article. By way of example, "an ing periosteum) by osteotomy, in 12 week old NZ element" means one element or more than one element. white rabbits, following a daily application for 7 days [0026] Throughout this specification and the claims to the epithelial surface at the fracture site of: Hibis- which follow, unless the context requires otherwise, the cus tiliaceus L precipitate of intermediate density in word "comprise", and variations such as "comprises" or ethanol (HeA Intermediate); Hibiscus tiliaceus L in 35 "comprising", will be understood to imply the inclusion of coconut oil following extraction with ethanol (eAo); a stated integer or step or group of integers or steps but Hibiscus tiliaceus L in ethanol (eA Ethanol); and a not the exclusion of any other integer or step or group of combination extract of Vigna marina (Burm.) Merr., integers or steps. Cocos nucifera L., and Terminalia catappa L. in co- [0027] Asused herein the terms "effectiveamount" and conut oil following extraction with ethanol (eNPKo). 40 "effective dose" include within their meaning a nontoxic but sufficient amount or dose of an extract or composition Figure 9 shows x-rays (A to D) and a CT scan (E) to provide the desired effect. The exact amount or dose of the spine of female New Zealand rabbits which required will vary from application to application and sub- underwent bilateral single level postero-lateral fu- ject to subject depending on factors such as whether the sion using autograft harvested from the iliac crest at 45 extract or composition is administered in vitro, ex vivo or 12 weeks old. Surgery was followed by routine care in vivo, the species being treated, the age and general (A and B) or daily application for 5 days and then condition ofthe subject, the severity of the condition being twice weekly application to 6 weeks to the epithelial treated, the particular extract or composition being ad- surface rostral to, but not overlying the fracture site ministered and the mode of administration and so forth. of an extract of Hibiscus tiliaceus L. in coconut oil 50 Thus, it is not possible to specify an exact effective following extraction in ethanol and hydrocarbon (C amount or dose. However, for any given case, an appro- to E). X-Rays show the bilateral spinal repair, with priate effective amount or dose may be determined by A the untreated and C the treated 6 week post op- one of ordinary skill in the art using only routine experi- erative healing fusion mass, and the donor harvest mentation. site on the iliac crest in untreated ( B) and treated ( D) 55 [0028] As used herein the term "extract" refers to an animals at 6 weeks. The CT scan shows the iliac active preparation derived from one or more plants. In crest of a treated animal at 6 weeks ( E). the context of the specification by "active" it is meant that the extract is capable of producing a desired therapeutic

4 7 EP 2 509 612 B1 8 benefit as disclosed herein. An extract is obtained by a pound to which it refers is suitable for use in contact with process of "extraction" which will be understood by those tissues of the body without undue toxicity, incompatibility, skilled in the art as, in general terms, comprising treating instability, irritation, allergic response, and the like, com- plant material with a solvent, a liquid, or a supercritical mensurate with a reasonable benefit/risk ratio. fluid to dissolve the active preparation and separate the 5 [0033] The present invention is predicated on the in- same from residual unwanted plant material. An extract ventors’ surprising finding, as exemplified herein, that the may be in liquid form (for example as a decoction, solu- use of extracts of various plants from Pacific islands pos- tion, infusion or tincture) or solid form (for example as a sess biological activity when administered to mammalian powder or granules). The term "combination extract" as skin, promoting bone and cartilage repair and the forma- used herein refers to an extract prepared from more than 10 tion of new bone and cartilage. one plant species. In a combination extract the plant ma- [0034] Accordingly, The invention is as defined in the terial from each of the plant species may be subjected claims. to the extraction process together or separately. That is, [0035] While exemplified using extracts ofHibiscus material from some or all of the species may be combined tiliaceus, it is also contemplated that the extract may also prior to addition of the solvent, liquid or supercritical fluid, 15 be derived from one or more of any Hibiscus sp. including and/or material from some or all of the species may be but not limited to, Hibiscus acetosella, Hibiscus bracken- independently treated with a solvent, liquid or supercrit- ridgei, Hibiscus cannabinus, Hibiscus diversifolius, Hi- ical fluid and the preparations so obtained are subse- biscus lavaterioide, Hibiscus ludwigii, Hibiscus macro- quently combined. As such, the same or different sol- phyllus, Hibiscus macropodus, Hibiscus elatus, Hibiscus vents (or liquids or supercritical fluids) may be used to 20 escobariae, Hibiscus ficulneus, Hibiscus paramutabilis, extract the active preparation from the different species. Hibiscus pedunculatus, Hibiscus platanifolius, Hibiscus The terms "extract" and "combination extract" may be radiatus, Hibiscus rosa-sinensis, Hibiscus sabdariffa, Hi- used interchangeably throughout the specification. biscus schizopetalus, Hibiscus scottii, Hibiscus socotra- [0029] The terms "promoting", "promotion" and varia- nus, Hibiscus sinosyriacus, Hibiscus splendens, Hibis- tions thereof as used in the context of bone or cartilage 25 cus stenanthus, Hibiscus striatus, Hibiscus syriacus, Hi- formation, repair and regeneration refer to the ability of biscus trilobus, or Hibiscus waimeae. an extract or composition as disclosed herein to induce, [0036] Extracts and compositions in accordance with stimulate, enhance, or otherwise effect or cause, either some embodiments may further comprise extracts of directly or indirectly, the formation of new bone or carti- Vigna sp. and/or Terminalia sp. In particular embodi- lage, or the repair or regeneration of bone or cartilage. 30 ments, the Vigna sp. is Vigna marina (Burm.) and/or the The promotion may occur in vivo, ex vivo or in vitro. The Terminalia sp. is Terminalia catappa. terms "enhancing", "enhancement" and variations there- [0037] It is also contemplated that the Vigna sp. extract of as used herein in the context of bone or cartilage for- may be derived from one or more of, but not limited to, mation, repair and regeneration refer to the ability of an Vigna aconitifolia, Vigna angularis, Vigna caracalla, extract or composition as disclosed herein to enhance or 35 Vigna debilis, Vigna dinteri, Vigna lanceolata, Vigna lute- increase, either directly or indirectly, the rate or degree ola, Vigna maritime, Vigna mungo, Vigna o-wahuensis, of action of natural physiological process(es) involved in Vigna parkeri, Vigna radiata, Vigna speciosa, Vigna sub- bone or cartilage formation, repair and/or regeneration. terranea, Vigna trilobata, Vigna umbellata, Vigna unguic- [0030] As used herein the term "subject" includes hu- ulata or Vigna vexillate. mans, primates, livestock animals (eg. sheep, pigs, cat- 40 [0038] It is also contemplated that the Terminalia sp. tle, horses, donkeys), laboratory test animals (eg. mice, extractmay be derived from one or moreof, but not limited rabbits, rats, guinea pigs), companion animals (eg. dogs, to, Terminalia acuminata, Terminalia alata, Terminalia cats), show animals (eg. dogs, cats, pigs, cattle, sheep, altissima, Terminalia amazonia, Terminalia angustifolia, horses) and captive wild animals (eg. foxes, kangaroos, Terminalia arborea, Terminalia arbuscula, Terminalia ar- deer). Typically, the subject is human or a laboratory test 45 chipelagi, Terminalia arjuna, Terminalia australis, Termi- animal. Even more typically, the subject is a human. nalia avicennioides, Terminalia bellirica, Terminalia bi- [0031] As used herein the terms "treating", "treatment", alata, Terminalia brachystemma, Terminalia brassii, Ter- "preventing" and "prevention" refer to any and all uses minalia bucidoides, Terminalia buceras (Bucida which remedy a condition or symptoms, prevent the es- buceras), Terminalia bursarina, Terminalia calamansa- tablishment of a condition or disease, or otherwise pre- 50 nai, Terminalia chebula, Terminalia cherrieri, Terminalia vent, hinder, retard, or reverse the progression of a con- ciliata, Terminalia citrina, Terminalia copelandii, Termi- dition or disease or other undesirable symptoms in any nalia corticosa, Terminalia eddowesii, Terminalia edulis, way whatsoever. Thus the terms "treating" and "prevent- Terminalia elliptica, Terminalia eriostachya, Terminalia ing" and the like are to be considered in their broadest erythrophylla, Terminalia ferdinandiana, Terminalia foe- context. For example, treatment does not necessarily im- 55 tidissima, Terminalia franchetii, Terminalia glabrescens, ply that a patient is treated until total recovery. Terminalia glaucifolia, Terminalia hararensis, Terminalia [0032] In the context of this specification, the term hecistocarpa, Terminalia intermedia, Terminalia ivoren- "pharmaceutically acceptable" means that the com- sis, Terminalia januariensis, Terminalia kaernbachii, Ter-

5 9 EP 2 509 612 B1 10 minalia kangeanensis, Terminalia kuhlmannii, Termina- ment(s) and extract or composition may also be cultured lia latifolia, Terminalia mantaly, Terminalia molinetii Ter- together with a bone fixation device or a prosthesis if minalia muelleri, Terminalia myriocarpa, Terminalia appropriate. Following culture under suitable conditions nitens, Terminalia novocaledonica, Terminalia oblonga- and for a suitable period of time, to at least initiate bone ta, Terminalia obovata, Terminalia oliveri, Terminalia5 formation, repair or regeneration, the bone fragment(s) paniculata, Terminalia parviflora, Terminalia pellucida, or bone segment(s), and optionally the bone fixation de- Terminalia phanerophlebia, Terminalia phellocarpa, Ter- vice or prosthesis may be reintroduced into the subject. minalia prunioides, Terminalia reitzii, Terminalia rerei, The culture conditions will depend on the application, Terminalia schimperiana, Terminalia sericea, Terminalia however suitable conditions can be readily determined seriocarpa, Terminalia subspathulata, Terminalia super- 10 by a person skilled in the art without the need for undue ba, Terminalia tripteroides or Terminalia volucris. experimentation. Alternatively, circumstances may ne- [0039] Also disclosed are methods for the promotion cessitate the use of bone fragment(s) or segment(s) from or enhancement of bone or cartilage formation, repair or other individuals or organisms. regeneration employing extracts and compositions as [0042] Similarly, osteoblasts or osteoblastic progenitor disclosed herein. Such methods find application in, inter 15 cells may be cultured ex vivo with an extract or compo- alia, the treatment of bone or cartilage injuries, defects sition as disclosed herein prior to their introduction into or diseases. The injury, defect or disease may be acute a subject in need of treatment. The cells may be autolo- or chronic. Those skilled in the art will readily appreciate gous (autogeneic), allogeneic or xenogeneic. Ex vivo cell the scope of injuries, defects and diseases to which the therapy may also be employed using mesenchymal stem embodiments disclosed herein relate, being those in20 cells taken from, for example, bone marrow or adult pe- which the formation of new bone or cartilage, the repair ripheral blood, embryonic stem cells, adult stem cells or of damaged or otherwise defective bone or cartilage, or any other multipotent, pluripotent or totipotent cells, or the regeneration of bone or cartilage is desirable or ad- ’designer’ cells generated in vitro. The term "osteoblastic vantageous. By way of example only, the injury may be, progenitor cells" as used herein encompasses all for example, the result of trauma such as a bone fracture 25 multipotent, pluripotent and totipotent cells, whether nat- or meniscal injury. By way of example, defects and dis- urally-derived or artificially created, that have the ability eases include congenital bone defects, bone or spinal to differentiate into osteoblasts. deformation, osteosarcoma, bone dysplasia, osteoporo- [0043] Extractsmay be aqueous, oil and/or organic sol- sis, osteomalacia (rickets), osteogenesis imperfecta vent based extracts, obtained by single, combined and/or (brittle bone disorder), Paget’s disease of the bone, os- 30 successiveextraction ofany available plant material such teoarthritis, and other diseases and conditions charac- as leaves, roots, bark, stems, fruits, shoots, nuts, husks terised by or associated with abnormal bone metabolism, of nuts, seeds, seed capsules, kernels, flowers, vine formation or resorption. and/or wood. Suitable extraction processes, and suitable [0040] Embodiments as disclosed herein also find ap- solvents and liquids for extraction are known to those plication in the context of surgical procedures to correct 35 skilled in the art. Aqueous solvents (for example water, bone injuries, deformations or diseases where the pro- acids, bases); oils (for example oil derived from Cocos motion or enhancement of bone growth or regeneration nucifera); and organic solvents, which can be polar (such is advantageous. For example, the embodiments dis- as alcohols for example ethanol), non-polar (for example closed herein may be used in conjunction with a skeletal hexane) and/or halogenated (for example dichlorometh- fusion (e.g. spinal fusion) procedure, spinal disc recon- 40 ane), used for extraction can either be used sequentially struction or removal, or the implantation of bone illingf for extraction or in combination mixture. Importantly, as material (such as hydroxyapatite blocks, demineralised exemplified herein, the activity of the extract is main- bon matrix plugs, collagen matrices), a fixation device tained when extracted into Cocos nucifera oil, polar sol- (such as a rod, screw, pin, plate or the like), surgical vents (e.g. ethanol) or non-polar solvents (e.g. isopen- implant or prosthetic device (such as a prosthetic hip or 45 tane). Supercritical fluid extraction using, for example, knee), or with a bone cement (such as that used to fill a supercritical nitrogen or carbon dioxide, may also be space between a bone and a prosthetic device or to an- used in accordance with the invention to obtain extracts. chor a prosthetic device to a bone). The implanted ma- [0044] Further, it will be appreciated by those skilled terial, implant or device may be resorbable or non-re- in the art that an extract of the invention may be subjected sorbable. 50 to one or more post extraction steps to, for example, in- [0041] In particular embodiments application of ex- crease or maintain the stability of the extract, modify or tracts and compositions for the purposes of treating bone change the physical form of the extract or assist in for- injuries, defects and disorders may be achieved using mulating the extract into a composition for administration ex vivo procedures. For example, in the case of injuries to a subject. By way of example only a liquid form extract such as bone fractures, a bone fragment(s) or bone seg- 55 may be lyophilised to produce a solid form of the extract. ment(s) may be removed from the subject to be treated [0045] Extracts may be derived from any suitable plant and cultured ex vivo with an extract or composition as material. Suitable plant material includes leaves, roots, disclosed herein. The bone fragment(s) or bone seg- bark, stems, fruits, shoots, nuts, husks of nuts, seeds,

6 11 EP 2 509 612 B1 12 seed capsules, kernels, flowers, vine or wood. The plant 0.001% and about 10%, or between about 0.005% and material may be, for example, fresh, dried or freeze dried. about 10%, or between about 0.01% and about 10%, or For any given plant species more than one plant material between about 0.05% and about 10%, or between about may be used for the production of extracts. Where de- 0.05% and about 5% in the topical composition. rived from Hibiscus tiliaceus L., typically the extract is a 5 [0050] Compositions as disclosed herein may be ad- leaf, vine and/or bean extract.. ministered via any convenient or suitable route such as [0046] Extract(s) may be administered in accordance for example by parenteral, topical, oral, or nasal routes. with the present invention in the form of pharmaceutical Parenteral administration may comprise, for example, in- compositions, which compositions may comprise one or traosseous infusion, or inthrathecal, intravenous, intraar- more pharmaceutically acceptable carriers, excipients or 10 terial, intramuscular, or subcutaneous administration. In diluents. Extracts may further be combined with other some embodiments, treatment may be effected using therapeutic agents for example, but not limited to, anti- methods and compositions as disclosed herein during biotics, antimicrobial agents, antiseptics, anaesthetics, arthroscopic or open surgical procedures. and/or other bone or cartilage growth or repair promoting [0051] Topical formulations typically comprise one or agents. 15 more extracts of the invention together with one or more [0047] It will be understood that the specific dose level acceptable carriers, and optionally any other therapeutic of a composition for use according to the invention for ingredients. Formulations suitable for topical administra- any particular individual will depend upon a variety of tion may be in any suitable form, formulated for example factors including, for example, the activity of the specific as liniments, lotions, creams, gels, ointments or pastes. extract(s) employed, the age, body weight, general20 Examples of pharmaceutically acceptable carriers or health and diet of the individual to be treated, the time of diluents are demineralised or distilled water; saline solu- administration, the stability of the extract(s), the site of tion; vegetable based oils such as peanut oil, safflower application on the body, and combination with any other oil, olive oil, cottonseed oil, maize oil, sesame oil, arachis treatment or therapy. Single or multiple administrations oil or coconut oil; silicone oils, including polysiloxanes, can be carried out with dose levels and pattern being25 such as methyl polysiloxane, phenyl polysiloxane and determined as required depending on the circumstances methylphenyl polysolpoxane; volatile silicones; mineral and the individual to be treated. Suitable dosage regimes oils such as liquid paraffin, soft paraffin or squalane; cel- can readily be determined by the skilled addressee. A lulose derivatives such as methyl cellulose, ethyl cellu- broad range of doses may be applicable. Considering a lose,carboxymethylcellulose, sodium carboxymethylcel- human subject, for example, from about 0.1 mg to about 30 lulose or hydroxypropylmethylcellulose; lower alkanols, 1 mg of extract may be administered per kilogram of body for example ethanol or iso-propanol; lower aralkanols; weight per day. Dosage regimens may be adjusted to lower polyalkylene glycols or lower alkylene glycols, for provide the optimum therapeutic response. For example, example polyethylene glycol, polypropylene glycol, eth- several divided doses may be administered hourly, daily, ylene glycol, propylene glycol, 1,3-butylene glycol or weekly, monthly or at other suitable time intervals or the 35 glycerin; fatty acid esters such as isopropyl palmitate, dose may be proportionally reduced as indicated by the isopropyl myristate or ethyl oleate; polyvinylpyrridone; exigencies of the situation. agar; carrageenan; gum tragacanth or gum acacia, and [0048] Generally, an effective dosage is expected to petroleum jelly. Typically, the carrier or carriers will form be in the range of about 0.0001 mg to about 1000 mg from 10% to 99.9% by weight of the compositions. per kg body weight per 24 hours; typically, about 0.001 40 [0052] Lotions include those suitable for application to mg to about 750 mg per kg body weight per 24 hours; the skin or to an epidermal appendage. Lotions or lini- about 0.01 mg to about 500 mg per kg body weight per ments for application to the skin may also include an 24 hours; about 0.1 mg to about 500 mg per kg body agent to hasten drying and to cool the skin, such as an weight per 24 hours; about 0.1 mg to about 250 mg per alcohol or acetone, and/or a moisturiser such as glycerol, kg body weight per 24 hours; about 1.0 mg to about 250 45 or oil such as coconut oil, castor oil or arachis oil. mg per kg body weight per 24 hours. More typically, an [0053] Creams, ointments or pastes are semi-solid for- effective dose range is expected to be in the range about mulations of the extract for external application. They 1.0 mg to about 200 mg per kg body weight per 24 hours; may be made by mixing the extract in finely-divided or about 1.0 mg to about 100 mg per kg body weight per 24 powdered form, alone or in solution or suspension in an hours; about 1.0 mg to about 50 mg per kg body weight 50 aqueous or non-aqueous fluid, with a greasy or non- per 24 hours; about 1.0 mg to about 25 mg per kg body greasy basis. The basis may comprise hydrocarbons weight per 24 hours; about 5.0 mg to about 50 mg per such as hard, soft or liquid paraffin, glycerol, beeswax, kg body weight per 24 hours; about 5.0 mg to about 20 a metallic soap; a mucilage; an oil of natural origin such mg per kg body weight per 24 hours; about 5.0 mg to as coconut, almond, corn, arachis, castor or olive oil; wool about 15 mg per kg body weight per 24 hours. 55 fat or its derivatives, or a fatty acid such as stearic or [0049] The extract may be present in an amount be- oleic acid together with an alcohol such as propylene tween about 0.001% (w/w) and about 15% (w/w), or be- glycol or macrogols. tween about 0.001% and about 12%, or between about [0054] The compositions may be included in topical

7 13 EP 2 509 612 B1 14 vehicles in an amount between about 0.001% (w/w) and freeze-drying technique which yield a powder of the ex- about 90% (w/w), or between about 1% (w/w) and about tract plus any additional desired extract from previously 50% (w/w), or between about 1% (w/w) and about 40% sterile-filtered solution thereof. (w/w), or between about 1% (w/w) and about 20% (w/w) [0058] The compositions may also be conveniently or about 0.01%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 5 presented in unit dosage form and prepared by any of 8%, 9%, 10%, 11%, 12%, 13% 14%, 15%, 16%, 17%, themethods well known in the art of pharmacy.The meth- 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, od may include the step of bringing the components of 60%, 65%, 70%, 75%, 80%, 85%, 90%. The extracts the oral composition into association with a carrier which and/or compositions also may be impregnated in any constitutes one or more accessory ingredients. In gen- form into transdermal patches, plasters, and dressings 10 eral, the compositions are prepared by uniformly and in- such as bandages or hydrocolloid dressings, for example timately bringing into association the components of the in liquid or semiliquid form. oral composition with a liquid carrier or finely divided solid [0055] The extracts and/or compositions may also be carrier, or both and then, if necessary, shaping the prod- coated on to or used as an adjunct to a bone fixation uct into the desired composition. device, a prosthesis or a bone cement. The extracts15 [0059] Compositions suitable for oral administration and/or compositions may also be used as an additive to may be presented as discrete units (i.e. dosage forms) a bone graft extender or bone substitute, for example, such as gelatine or HPMC capsules, cachets or tablets, allograft bone, hydroxyapatite, wollanosite or tricalcium each containing a predetermined amount each compo- phosphate. nent of the composition as a powder, granules, as a so- [0056] In particular circumstances, for example in the 20 lution or a suspension in an aqueous liquid or a non- post surgical promotion of bone and cartilage formation, aqueous liquid, or as an oil-in-water liquid emulsion or a repair or regeneration, administration of compositions by water-in-oil liquid emulsion. parenteral administration, typically injection, may be ap- [0060] When the composition is formulated as cap- propriate. Pharmaceutical forms suitable for injectable sules, the components of the oral composition may be use include sterile aqueous solutions (where water sol- 25 formulated with one or more pharmaceutically accepta- uble) or dispersions and sterile powders for the extem- ble carriers such as starch, lactose, microcrystalline cel- poraneous preparation of sterile injectable solutions. It lulose and/or silicon dioxide. Additional ingredients may must be stable under the conditions of manufacture and include lubricants such as magnesium stearate and/or storage and must be preserved against the contaminat- calcium stearate. ing action of microorganisms such as bacteria and fungi. 30 [0061] Tablets may be prepared by compression or The carrier can be a solvent or dispersion medium con- moulding, optionally with one or more accessory ingre- taining, for example, water, ethanol, polyol (for example, dients. Compressed tablets may be prepared by com- glycerol, propylene glycol and liquid polyethylene glycol, pressing in a suitable machine the components of the and the like), suitable mixtures thereof, and vegetable oral composition in a free-flowing form such as a powder oils. The proper fluidity can be maintained, for example, 35 or granules, optionally mixed with a binder, lubricant (for by the use of a coating such as lecithin, by the mainte- example magnesium stearate or calcium stearate), inert nance of the required particle size in the case of disper- diluentor asurface active/dispersing agent. Moulded tab- sion and by the use of superfactants. The preventions of lets may be made by moulding a mixture of the powdered the action of microorganisms can be brought about by composition moistened with an inert liquid diluent, in a various antibacterial and antifungal agents, for example, 40 suitable machine. The tablets may optionally be coated, parabens, chlorobutanol, phenol, sorbic acid, thimerosal for example, with an enteric coating and may be formu- and the like. In many cases, it will be preferable to include lated so as to provide slow or controlled release of the isotonic agents, for example, sugars or sodium chloride. composition therein. Prolonged absorption of the injectable compositions can [0062] Are disclosed herein combination therapies, be brought about by the use in the compositions of agents 45 wherein extracts or compositions as described herein are delaying absorption, for example, aluminum monostea- coadministered with other suitable agents or treatments rate and gelatin. which may facilitate the desired therapeutic effect. For [0057] Sterile injectable solutions are prepared by in- example, one may seek to administer antibiotics, antimi- corporating the active compounds in the required amount crobial agents, anaesthetics, analgesics, or other bone in the appropriate solvent with various of the other ingre- 50 or cartilage growth- or repair-promoting agents in com- dients enumerated above, as required, followed by fil- bination with extracts or compositions disclosed herein. tered sterilisation. Generally, dispersions are prepared By "coadministered" is meant simultaneous administra- by incorporating the various sterilised extract(s) into a tion in the same formulation or in two different formula- sterile vehicle which contains the basic dispersion medi- tions via the same or different routes or sequential ad- um and the required other ingredients from those enu- 55 ministration by the same or different routes. By "sequen- merated above. In the case of sterile powders for the tial" administration is meant a time difference of from sec- preparation of sterile injectable solutions, the preferred onds, minutes, hours or days between the administration methods of preparation are vacuum drying and the of the two types of agents. The agents may be adminis-

8 15 EP 2 509 612 B1 16 tered in any order. For example, is disclosed the admin- er was then depressed thereby extracting the oil. The istration of a lipid or an oil, for example Cocos nucifera mixture was left to settle for a further 5 min and the plung- oil prior to, or after, the extract or composition is admin- er depressed again to release more oil. This process was istered. repeated 5 times until no further oil was obtained. When [0063] The reference in this specification to any prior 5 the extract was prepared by heating, Hibiscus the publication (or information derived from it), or to any mat- tiliaceus L. and coconut oil mixture was heated in a cru- ter which is known, is not, and should not be taken as an cible in a water bath at 100°C for 20 min, removed from acknowledgment or admission or any form of suggestion the heat, and then immediately filtered and pressed to that that prior publication (or information derived from it) extract the coconut oil. Any remaining moisture or solid or known matterforms part of the common generalknowl- 10 in the extract after cold pressing or heating and filtering edge in the field of endeavour to which this specification were removed by inverting the coconut oil extract and relates. The present invention will now be described with storing it at less than 20°C until the coconut oil solidified reference to the following specific examples, which and then decanting the solidified coconut oil; or by de- should not be construed as in any way limiting the scope canting in a separating funnel. of the invention. 15 2.2 Hibiscus tiliaceus L. extract in coconut oil following Examples extraction in ethanol

Example 1 - Preparation of a combination extract of [0067] The fresh crushed bark of Hibiscus tiliaceus L. Vigna marina (Burm.) Merr., Cocos nucifera L., Ter- 20 (1000 g) were immersed in 95-100% ethanol (2000 mL) minalia catappa L. and Hibiscus tiliaceus L. in coco- and left to steep for 6 h. The ethanol was then removed nut oil and the Hibiscus tiliaceus L. steeped in a second aliquot of ethanol (2000 mL) for a further 6 h. The resultant eth- [0064] Coconut oil (200 mL) was added to fresh shred- anol extracts were added to coconut oil (250 mL). More ded leaves of Vigna marina (Burm.) Merr. (100 g) and 25 than half the volume of ethanol was removed by heating fresh shredded leaves of Terminalia catappa L. (100 g). above the boiling point of ethanol. The coconut oil was The mixture was placed in a crucible in a water bath at then extracted by decanting from the solution following 100°C for 20 min. The mixture was removed from the standing for at least 2 h in a decanting vessel; or by in- heat and immediately filtered and pressed to extract the verting the coconut oil extract and storing it at less than coconut oil. The resultant coconut oil, containing extracts 30 20°C until the coconut oil solidified and then decanting of Vigna marina (Burm.) Merr. and Terminalia catappa the solidified coconut oil. L., was added to the fresh crushed husk of a green nut of Cocos nucifera L. (100 g) and the shaved bark of Hi- 2.3 Hibiscus tiliaceus L. extract in coconut oil following biscus tiliaceus L. (100 g). The mixture was left to settle extraction in ethanol and hydrocarbon for 4 h and then filtered and pressed to extract the coconut 35 oil. [0068] The fresh crushed bark of Hibiscus tiliaceus L. [0065] The coconut oil was then inverted and stored (1000 g) were immersed in 95-100% ethanol (2000 mL) at less than 20°C until the coconut oil solidified. Any re- and left to steep for 6 h. The ethanol was then removed maining moisture or solid in the mixture were removed and the Hibiscus tiliaceus L. steeped in a second aliquot by decanting from the solidified coconut oil. The coconut 40 of ethanol (2000 mL) for a further 6 h. oil was then heated in a hot water bath at approximately [0069] The resultant ethanol solution was agitated with 56°C and filtered. The resultant filtrate contained a com- hexane, pentane, methyl butane or an equivalent hydro- bination extract of Vigna marina (Burm.) Merr., Cocos carbon or fluoro/chloro/bromo-hydrocarbon (250 mL). nucifera L., Terminalia catappa L. and Hibiscus tiliaceus Thehydrocarbon layer andoily foam layerwere decanted L. 45 from the solution following standing for at least 2 h in a decanting vessel; or by the dilution of the ethanol solution Example 2 - Preparation of extracts of Hibiscus from 95% to a lower concentration, nominally but not nec- tiliaceus L. essarily 50% ethanol and then decanted after settling of the layers. Water (250 mL) or alcohol (250 mL) was add- 2.1 Hibiscus tiliaceus L. extract in coconut oil 50 ed to the extracted hydrocarbon layer. To this coconut oil (250 mL) was added and the mixture agitated and [0066] The fresh crushed bark of Hibiscus tiliaceus L. allowed to settle. The hydrocarbon was removed from (100 g) were immersed in coconut oil (100 mL). The ex- heating the mixture to the boiling point of the hydrocar- tract was prepared by cold pressing the Hibiscus tiliaceus bon. L. and coconut oil mixture; or by heating the mixture. The 55 cold pressing was carried out by placing theHibiscus tiliaceus L. and coconut oil mixture inside a new coffee plunger and leaving the mixture to settle for 1 h, the plung-

9 17 EP 2 509 612 B1 18

2.4 Hibiscus tiliaceus L. extract in coconut oil following Example 4 - Treatment of fractures in oestrogen and extraction in ethanol non-oestrogen deficient bones

[0070] The fresh crushed bark of Hibiscus tiliaceus L. [0075] A study was carried out to assess the effect of (1000 g) were immersed in 95-100% ethanol (2000 mL) 5 an extract of Hibiscus tiliaceus L. prepared as described and left to steep for 6 h. The ethanol was then removed in Example 2.1, on bone fracture repair in oestrogen de- and the Vigna marina (Burm.) Merr. steeped in a second ficient and non-oestrogen deficient rats. In the study a aliquot ofethanol (2000 mL) for a further 6 h.The resultant daily dose of 1 mL of the extract was applied topically to ethanol solution was agitated with coconut oil (250 mL). the epithelial surface of 18 month old, oestrogen deficient The hydrocarbon was decanted from the solution follow- 10 (ovaries removed at age 6 weeks) rats and 18 month old ing standing for at least 2 h in a decanting vessel; or by rats with their ovaries intact (a control surgical procedure the dilution of the ethanol solution from 95% to a lower was carried out at age 6 weeks and the rats allowed to concentration, nominallybut notnecessarily 50% ethanol recover), following a surgically created fracture of the fe- and then decanted after settling of the layers. The de- mur by osteotomy, and operative repair with internal fix- canting of the coconut oil may be facilitated by the addi- 15 ation. The extract was applied daily to the test animals tion of a low boiling point hydrocarbon to the mixture as for a total of 3 weeks. Control animals were 18 month it agitates and settles. The coconut oil was then inverted old, oestrogen deficient (ovaries removed at age 6 and stored at less than 20°C until the coconut oil solidi- weeks) rats and 18 month old rats with their ovaries intact fied, or by decanting in s separation vessels. with no topical application of the extract. At 3 weeks the 20 histology of the animals was assessed, with particular Example 3 - Effect of individual extracts of Hibiscus attention paid to the cartilage and new bone formation in tiliaceus L. on skin the healing fracture. [0076] Figures 3A to 3H show the histology at the frac- [0071] An assessment of the activity of the extracts ture site. The results indicate extensive new cartilage prepared as described in Example 2 on skin was carried 25 growth and new bone formation following the cartilage out. A daily dose of 1 mL of Hibiscus tiliaceus L. extract caps in the treated groups, particularly of note is the in coconut oil (Example 2.1) and 1 mL Hibiscus of amount occurring in the oestrogen deficient bones. tiliaceus L extract in ethanol (Example 2.2). The extracts [0077] Figure 4 is a graph showing histological scoring were applied topically to the epithelial surface of the of the fracture sites of untreated oestrogen deficient rats backs of 10-14 week old female rats. The controls were 30 (Ovx Control) and treated oestrogen deficient rats (Ovx 10-14 week old female rats treated daily with 1 mL of rx) rats. In order to determine the total histological scoring coconut oil or left untreated. the following fracture healing and tissue response pa- [0072] Figures 1A to 1D show the histology of the skin rameters were scored: callous formation, bone union, at 7 days of the untreated control (1A) after a daily ap- marrow changes and cortex remodelling. Each parame- plication of coconut oil (1B); an extract preparation of 35 ter was scored from 0 (no callous formation; no new bone Hibiscus tiliaceus L in ethanol (1C); and coconut oil fol- in the fracture; fibrous tissue or red; and no remodelling) lowing extraction with ethanol (1D). The histological pro- to 3 (full callous formation across the defect; full bone files show that the extract has limited activity in ethanol, bridge union; adult type fatty marrow; and full remodelling but that in coconut oil the extract induces hypertrophy of cortex), with a score of 1 indicating mild healing (<50%) the epithelium and the epithelial appendages in particular 40 and a score of 2 indicating moderate healing (>50% but hair follicles. less than full fracture healing or tissue response). After [0073] An assessment of the activity of the extracts 3 weeks there was a significantly higher total histological prepared as described in Examples 1 and 2.2 on skin score for the fracture healing and tissue response at the was carried out. A daily dose of 1 mL of the extracts were fracture sites of treated oestrogen deficient rats com- applied topically to the epithelial surface of the backs of 45 pared to untreated oestrogen deficient rats. 12 week old female New Zealand rabbits. The control was the untreated skin of 12 week old female New Zea- Example 5 - Treatment of fractures land rabbits. [0074] Figures 2A to 2F show the histology of the skin [0078] A study was carried out to assess the effect of at 7 days of the untreated control (2A and B); and after 50 an extract of Hibiscus tiliaceus L. prepared as described a daily application of a combination extract of Vigna main Examples 1 and 2.1 on bone fracture healing. In the rina (Burm.) Merr., Cocos nucifera L., Terminalia catappa study a daily dose of 1 mL of the extract was applied L. and Hibiscus tiliaceus L. in coconut oil prepared as topically to the epithelial surface of 12 week old NZ white described in Example 1 (2C and D); Hibiscus and rabbits, following a surgically created 20 mm defect of tiliaceus L in coconut oil prepared as described in as Ex- 55 the ulna (including periosteum) by osteotomy, and with ample 2.2 (2E and F). The histological profiles show that no graft or substitute filler. The extract was applied daily the extracts induce hypertrophy of the epithelium and the to the test animals for a total of 1 week. Control animals epithelial appendages in particular hair follicles. were 12 week old NZ white rabbits with no topical appli-

10 19 EP 2 509 612 B1 20 cation of the extract. At 1 week radiology and histology and Eosin covering the entire spinal fusion mass. The of the animals was assessed, with particular attention transverse processes can be seen at each end of the paid to cartilage and new bone formation. mass with the well formed marrow spaces and dense [0079] Figures 5A to 5D are radiographs of the bone cortical bone (A) and adjacent to the transverse process and Figures 6A to 6F and 7A to 7D show histology of the 5 is a small amount of cartilage (B). The bone fragments bone defect at the fracture site. The figures illustrate the from the autograft are still visible and are necrotic, with effect of the extracts of Hibiscustiliaceus L. on the healing the loss of lacunae (C). The centre of the fusion mass is of the fracture. After seven days there was significantly predominantly fibrous tissue (D) and some new woven more bone formation, new cartilage formation and great- bone (E). er vascularity at the fracture site than in untreated or con- 10 [0084] Figure 10B shows the histology of the treated trol treatment fractions. autograft at 6 weeks, stained with Haematoxylin and [0080] Figure 8 is a graph of the bone growth score Eosin covering the entire spinal fusion mass. The trans- which shows the bone healing of the defect evaluated verse processes can be seen at each end of the mass using a healing score definition based on the percent of with the well formed marrow spaces and dense cortical bone or cartilage occupied space. The following scoring 15 bone (A) and adjacent to the transverse process is cor- system was used: 0 = no visible bone; 1 = minimal (1% ticated new bone (B). The bone fragments from the to 20%) new bone growth no cartilage; 2 = mild (1 to autograft are barely visible and are mostly absorbed and 20%) cartilage with new bone growth; 3 = moderate (20% replaced (C). The centre of the fusion mass is predomi- to 40%) cartilage and bone growth; 4 = marked (60 to nantly cartilage (D), new marrow spaces (E) and some 80%) cartilage and bone growth; 5 = complete defect 20 new woven bone (F). filled with new bone and cartilage; 6 = defect filled with [0085] Figure11 showthe results of mechanical testing new bone and no cartilage; 7 = cortical bone replacing of the treated autograft at 8 weeks compared to untreated woven bone; and 8 = new marrow space forming. After controls at 12 weeks. The ligaments and inter-vertebral 7 days there was significantly more bone formation at discs were surgically cut in all spines leaving the trans- the fracture site treated with Hibiscus tiliaceus L in coco- 25 verse process spinal fusion mass as the bridge between nut oil following extraction with ethanol (eAo) than the the vertebral bodies. Loading was applied until the fusion fracture sites treated with Hibiscus tiliaceus L precipitate mass broke. The peak load was recorded for each sam- of intermediate density in ethanol (HeA Intermediate) in ple. The data shown in Figure 11 suggests that the 8 untreated or control treatment fractions;Hibiscus week treated group is at least as strong as the 12 week tiliaceus L in ethanol (eA Ethanol); or a combination ex- 30 control with a trend towards greater bone strength. tract of Vigna marina (Burm.) Merr., Cocos nucifera L., and Terminalia catappa L. in coconut oil following extrac- Example 7 - Analysis of extracts of Hibiscus tiliaceus tion with ethanol (eNPKo). L.

Example 6 - Bone formation following a spinal fusion 35 [0086] A method of investigating the composition of auotgraft the extracts was developed using gas chromatographic- mass spectrometric (GC-MS) analysis. For example, [0081] A study was carried out to assess the effect of GC-MS analysis was carried out on an extract of Hibiscus an extract of Hibiscus tiliaceus L. prepared as described tiliaceus L. in coconut oil following extraction in ethanol in Example 2.4 on bone fracture repair. In the study a 40 and hydrocarbon (Example 2.3) and the hydrocarbon in- daily dose 1 mL of the extract was applied topically to termediate layer. the epithelial surface of 12 week old NZ white rabbits, [0087] Figure 12A shows mass chromatograms of an following anaesthetisation and a bilateral single level extract of Hibiscus tiliaceus L. in coconut oil with a main postero-lateral fusion using autograft harvested from the peak at 30.99 min. The intermediate layer also contained iliac crests. The extract (5 mL) was applied daily to the 45 a peak at 30.99 min (Figure 12B), however, the peak was test animals for 5 days and then twice weekly until sac- at least seven times less than that observed in the extract rifice. The control animals were 12 week old NZ white of Hibiscus tiliaceus L. in coconut oil. In summary, the rabbits with no topical application of the extract. dominant peaks in the mass chromatograms of the ex- [0082] Figures 9A to 9E show x-rays and a CT scan of tract of Hibiscus tiliaceus L. in coconut oil were at 30.99, the spine in treated and untreated controls. The Figures 50 35.47, 37.48, 39.56, 42.85, 43.28, 49.04, 52.51, 55.97 show the effect of the extracts of Hibiscus tiliaceus L. on and 58.54 min. The majority of the smaller peaks corre- bone growth surrounding the site of the autograft. After spond to ethyl esters of fatty acids found in coconut oil. only seven days there was more bone formation, new cartilage formation and greater vascularity at the fracture site than in untreated or control treatment fractures. 55 Claims [0083] Figures 10A and 10B show the histology of the spine at 6 weeks. Figure 10A shows the histology of the 1. A biologically active extract of Hibiscus tiliaceus L., control autograft at 6 weeks, stained with Haematoxylin wherein the extract is prepared using and/or com-

11 21 EP 2 509 612 B1 22

prises Cocos nucifera oil, a composition comprising progenitor cells, bone segments or fragments for use said biologically active extract and one or more phar- as claimed in any one of claims 1 to 6, wherein said maceutically acceptable carriers, diluents and/or ex- therapeutic use in the promotion is the promotion of cipients, or osteoblasts, osteoblastic progenitor bone or cartilage growth surrounding a bone or car- cells, bone segments or fragments cultured in the 5 tilage graft or implantable device. presence of said extract or composition for use in the treatment of bone or cartilage injury, defect or 11. An extract, composition, or osteoblasts, osteoblastic disease and/or for therapeutic use in the promotion progenitor cells, bone segments or fragments for use of bone or cartilage formation, repair or regeneration. as claimed in claim 10, wherein the implantable de- 10 vice is a bone fixation device or a prosthesis. 2. An extract, composition, osteoblasts, osteoblastic progenitor cells, bone segments or fragments, for 12. An extract, composition, or osteoblasts, osteoblastic use as claimed in claim 1, wherein theHibiscus progenitor cells, bone segments or fragments for use tiliaceus L extract is derived from one or more of a as claimed in claim 11, wherein the extract, the com- bark, leaf, vine, bean, husk or nut. 15 position, or the osteoblasts, osteoblastic progenitor cells or bone segments or fragments are coated on 3. An extract, composition, osteoblasts, osteoblastic to the prosthesis, used as an adjunct to the bone progenitor cells, bone segments or fragments, for fixation device, or used as an adjunct to a bone ce- use as claimed in claim 1 or claim 2, wherein the ment used to anchor the prosthesis or bone fixation Hibiscus tiliaceus L. extract is derived from bark. 20 device.

4. An extract, composition, osteoblasts, osteoblastic 13. Osteoblasts, osteoblastic progenitor cells or bone progenitor cells, bone segments or fragments, for segments or fragments for use in the formation or use as claimed in any one of claims 1 to 3, wherein growth of bone, wherein the osteoblasts, osteoblas- the Cocos nucifera oil is virgin Cocos nucifera oil, 25 tic progenitor cells, bone segments or fragments are refined Cocos nucifera oil, hydrogenated Cocos incubated in the presence of an effective amount of nucifera oil or fractionated Cocos nucifera oil. an extract or composition of any one of claims 1 to 4.

5. A composition for use as claimed in any one of claims 1-4, wherein the composition is administered topi- 30 Patentansprüche cally or parenterally. 1. Biologisch aktiver Extrakt von Hibiscus tiliaceus L., 6. A composition for use as claimed in any one of claims wobei der Extrakt präpariert wird mit Cocos nucifera- 1 to 5, wherein the composition further comprises an ÖI und/oder dieses umfasst, eine diesen biologisch extract of one or more of Vigna marina (Burm.) Merr., 35 aktiven Extrakt und mindestens einen pharmazeu- Cocos nucifera L., or Terminalia catappa L. tisch unbedenklichen Träger, Verdünnungsmittel und/oder Hilfsstoff umfassende Zusammensetzung, 7. An extract, composition, osteoblasts, osteoblastic oder Osteoblasten, osteoblastische Vorläuferzellen, progenitor cells, bone segments or fragments for use Knochensegmente oder -fragmente, die in Gegen- as claimed in any one of claims 1 to 6, wherein the 40 wart des Extraktes oder der Zusammensetzung ge- injury is a bone fracture. züchtet werden, zur Verwendung bei der Behand- lung einer Verletzung, eines Defektes und oder einer 8. An extract, composition, osteoblasts, osteoblastic Krankheit von Knochen oder Knorpel und/oder zur progenitor cells, bone segments or fragments for use therapeutischen Verwendung bei der Förderung der as claimed in any one of claims 1 to 6, wherein the 45 Bildung, Reparatur oder Regeneration von Knochen disease is a degenerative bone disease, optionally oder Knorpel. osteoporosis. 2. Extrakt, Zusammensetzung, Osteoblasten, osteo- 9. An extract, composition, osteoblasts, osteoblastic blastische Vorläuferzellen, Knochensegmente oder progenitor cells, bone segments or fragments for use 50 -fragmente zurVerwendung nach Anspruch1, wobei as claimed in any one of claims 1 to 6, wherein said der Hibiscus tiliaceus L.-Extrakt aus mindestens ei- therapeutic use in the promotion of bone or cartilage nem von Rinde, Blatt, Rebe, Bohne, Schale oder formation, repair or regeneration is in a subject un- Nuss abgeleitet ist. dergoing or that has undergone a bone or cartilage graft procedure, wherein optionally the graft is a skel- 55 3. Extrakt, Zusammensetzung, Osteoblasten, osteo- etal fusion. blastische Vorläuferzellen, Knochensegmente oder -fragmente zur Verwendung nach Anspruch 1 oder 10. An extract, composition, or osteoblasts, osteoblastic 2, wobei der Hibiscus tiliaceus L.-Extrakt aus der

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Rinde abgeleitet ist. delt.

4. Extrakt, Zusammensetzung, Osteoblasten, osteo- 12. Extrakt, Zusammensetzung, Osteoblasten, osteoblastische Vorläuferzellen, Knochensegmente oder blastische Vorläuferzellen, Knochensegmente oder -fragmente zur Verwendung nach einem der Ansprü- 5 -fragmente zur Verwendung nach Anspruch 11, wo- che 1 - 3, wobei das Cocos nucifera-ÖI Cocos nuci- bei der Extrakt, die Zusammensetzung, Osteoblas- fera-Jungernöl, verfeinertes Cocos nucifera-ÖI, hy- ten, osteoblastischen Vorläuferzellen, Knochenseg- driertes Cocos nucifera-ÖI oder fraktioniertes Cocos mente oder -fragmente als Beschichtung auf der nucifera-ÖI ist. Prothese vorliegen, als Hilfsmittel der Knochenfixa- 10 tionsvorrichtung verwendet oder als Hilfsmittel des 5. Zusammensetzung zurVerwendung nacheinem der zur Verankerung der Prothese bzw. Knochenfixati- Ansprüche 1 - 4, wobei die Zusammensetzung to- onsvorrichtung verwendeten Knochenzements ver- pisch oder parenteral verabreicht wird. wendet werden.

6. Zusammensetzung zurVerwendung nacheinem der 15 13. Osteoblasten, osteoblastische Vorläuferzellen oder Ansprüche1 - 5, wobei dieZusammensetzung ferner Knochensegmente oder - fragmente zur Verwen- einen Extrakt aus mindestens einem von Vigna ma- dung bei der Knochenbildung bzw. dem Knochen- rina (Burm.)Merr., Cocosnucifera L. oder Terminalia wachstum, wobei die Osteoblasten, osteoblasti- catappa L. umfasst. schen Vorläuferzellen, Knochensegmente oder 20 -fragmente in Gegenwart einer wirksamen Menge 7. Extrakt, Zusammensetzung, Osteoblasten, osteo- eines Extraktes bzw. einer Zusammensetzung nach blastische Vorläuferzellen, Knochensegmente oder einem der Ansprüche 1 - 4 inkubiert werden. -fragmente zur Verwendung nach einem der Ansprü- che 1 - 6, wobei es sich bei der Verletzung um eine Knochenfraktur handelt. 25 Revendications

8. Extrakt, Zusammensetzung, Osteoblasten, osteo- 1. Extrait biologiquement actif d’Hibiscus tiliaceus L., blastische Vorläuferzellen, Knochensegmente oder où l’extrait est préparé en utilisant et/ou comprend -fragmente zur Verwendung nach einem der Ansprü- de l’huile de Cocos nucifera, composition compre- che 1 - 6, wobei es sich bei der Verletzung um eine 30 nant ledit extrait biologiquement actif et un ou plu- osteodegenerative Krankheit, wahlweise Osteopo- sieurs véhicules, diluants et/ou excipients pharma- rose, handelt. ceutiquement acceptables, ou ostéoblastes, cellules progénitrices ostéoblastiques, segments ou frag- 9. Extrakt, Zusammensetzung, Osteoblasten, osteo- ments osseux mis en culture en présence dudit ex- blastische Vorläuferzellen, Knochensegmente oder 35 trait ou de ladite composition, pour une utilisation -fragmente zur Verwendung nach einem der Ansprü- dans le traitement d’une lésion, d’un défaut ou d’une che 1 - 6, wobei die therapeutische Verwendung bei maladie des os ou du cartilage et/ou pour une utili- der Förderung der Bildung, Reparatur oder Regene- sation thérapeutique dans la promotion de la forma- ration von Knochen oder Knorpel bei einem Proban- tion, la réparation ou la régénération d’os ou de car- den erfolgt, der einer Knochen- oder Knorpeltrans- 40 tilage. plantation unterzogen wird bzw. unterzogen worden ist, wobei es sich bei der Transplantation wahlweise 2. Extrait, composition, ostéoblastes, cellules progéni- um eine Skelettfusion handelt. trices ostéoblastiques, segments ou fragments osseux, pour une utilisation selon la revendication 1, 10. Extrakt, Zusammensetzung, Osteoblasten, osteo- 45 où l’extrait d’Hibiscus tiliaceus L. est dérivé d’un ou blastische Vorläuferzellen, Knochensegmente oder de plusieurs éléments parmi une écorce, une feuille, -fragmente zur Verwendung nach einem der Ansprü- une liane, un germe, une coque ou une noix. che 1 - 6, wobei es sich bei der therapeutischen Ver- wendung bei der Förderung um eine Förderung des 3. Extrait, composition, ostéoblastes, cellules progéni- Knochen-oder Knorpelwachstumsin der Umgebung 50 trices ostéoblastiques, segments ou fragments os- eines Knochen- oder Knorpeltransplantats oder ei- seux, pour une utilisation selon la revendication 1 ou nes implantierbaren Geräts handelt. la revendication 2, où l’extrait d’ Hibiscus tiliaceus L. est dérivé d’écorce. 11. Extrakt, Zusammensetzung, Osteoblasten, osteoblastische Vorläuferzellen, Knochensegmente oder 55 4. Extrait, composition, ostéoblastes, cellules progéni- -fragmente zur Verwendung nach Anspruch 10, wo- trices ostéoblastiques, segments ou fragments os- bei es sich beim implantierbaren Gerät um eine Kno- seux, pour une utilisation selon l’une quelconque des chenfixationsvorrichtung oder eine Prothese han- revendications 1 à 3, où l’huile deCocos nucifera

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est de l’huile de Cocos nucifera vierge, de l’huile de ou fragments osseux sont revêtus sur la prothèse, Cocos nucifera raffinée, de l’huile de Cocos nucifera utilisé(es) en tant que complément au dispositif de hydrogénée ou de l’huile de Cocos nucifera fraction- fixation osseuse, ou utilisé(es) en tant que complé- née. ment à un ciment osseux utilisé pour ancrer la pro- 5 thèse ou le dispositif de fixation osseuse. 5. Composition destinée à être utilisée selon l’une quelconque des revendications 1 à 4, où la composition 13. Ostéoblastes, cellules progénitrices ostéoblastiques est administrée par voie topique ou par voie paren- ou segments ou fragments osseux pour une utilisa- térale. tion dans la formation ou la croissance d’os, où les 10 ostéoblastes, cellules progénitrices ostéoblasti- 6. Composition destinée à être utilisée selon l’une quel- ques, segments ou fragments osseux sont in- conque des revendications 1 à 5, où la composition cubé(es) en présence d’une quantité efficace d’un comprend en outre un extrait d’un ou de plusieurs extrait ou d’une composition selon l’une quelconque de Vigna marina (Burm.) Merr., Cocos nucifera L., des revendications 1 à 4. ou Terminalia catappa L. 15

7. Extrait, composition, ostéoblastes, cellules progéni- trices ostéoblastiques, segments ou fragments osseux pour une utilisation selon l’une quelconque des revendications 1 à 6, où la lésion est une fracture 20 osseuse.

8. Extrait, composition, ostéoblastes, cellules progéni- trices ostéoblastiques, segments ou fragments osseux pour une utilisation selon l’une quelconque des 25 revendications 1 à 6, où la maladie est une maladie osseuse dégénérative, facultativement l’ostéoporo- se.

9. Extrait, composition, ostéoblastes, cellules progéni- 30 trices ostéoblastiques, segments ou fragments osseux pour une utilisation selon l’une quelconque des revendications 1 à 6, où ladite utilisation thérapeuti- que dans la promotion de la formation, la réparation ou la régénération d’os ou de cartilage est chez un 35 sujet subissant ou qui a subi une procédure de greffe d’os ou de cartilage, où facultativement la greffe est une arthrodèse squelettique.

10. Extrait, composition, ou ostéoblastes, cellules pro- 40 génitrices ostéoblastiques, segments ou fragments osseux pour une utilisation selon l’une quelconque des revendications 1 à 6, où ladite utilisation théra- peutique dans la promotion est la promotion de la croissance d’os ou de cartilage entourant une greffe 45 d’os ou de cartilage ou un dispositif implantable.

11. Extrait, composition, ou ostéoblastes, cellules pro- génitrices ostéoblastiques, segments ou fragments osseux pour une utilisation selon la revendication 50 10, où le dispositif implantable est un dispositif de fixation osseuse ou une prothèse.

12. Extrait, composition, ou ostéoblastes, cellules pro- génitrices ostéoblastiques, segments ou fragments 55 osseux pour une utilisation selon la revendication 11, où l’extrait, la composition, ou les ostéoblastes, cellules progénitrices ostéoblastiques ou segments

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REFERENCES CITED IN THE DESCRIPTION

This list of references cited by the applicant is for the reader’s convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard.

Patent documents cited in the description

• US 2009155216 A [0008]

Non-patent literature cited in the description

• Traditional and herbal medicine in the cook islands. • SMITH, A.C. Flora Vitiensis Nova: A new Flora of Fiji WHISTLER et al. Journal of Ethnopharmacology. El- (Spermatophytes Only). Pacific Tropical Botanical sevier Scientific Publishers Ltd, 01 July 1985, vol. 13 Garden, 1981, vol. 2, 417 [0007] [0006]