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WO1999018991A1 - Materiaux et methodes relatives a la reparation du cartilage - Google Patents

Materiaux et methodes relatives a la reparation du cartilage Download PDF

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Publication number
WO1999018991A1
WO1999018991A1 PCT/GB1998/003089 GB9803089W WO9918991A1 WO 1999018991 A1 WO1999018991 A1 WO 1999018991A1 GB 9803089 W GB9803089 W GB 9803089W WO 9918991 A1 WO9918991 A1 WO 9918991A1
Authority
WO
WIPO (PCT)
Prior art keywords
use according
agent
cartilage
factor
medicament
Prior art date
Application number
PCT/GB1998/003089
Other languages
English (en)
Inventor
Charles William Archer
Carl Ralph Flannery
Original Assignee
Smith & Nephew Plc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Smith & Nephew Plc filed Critical Smith & Nephew Plc
Priority to AU94512/98A priority Critical patent/AU9451298A/en
Publication of WO1999018991A1 publication Critical patent/WO1999018991A1/fr

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/185—Nerve growth factor [NGF]; Brain derived neurotrophic factor [BDNF]; Ciliary neurotrophic factor [CNTF]; Glial derived neurotrophic factor [GDNF]; Neurotrophins, e.g. NT-3
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1858—Platelet-derived growth factor [PDGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1875—Bone morphogenic factor; Osteogenins; Osteogenic factor; Bone-inducing factor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • A61K38/22—Hormones
    • A61K38/30—Insulin-like growth factors, i.e. somatomedins, e.g. IGF-1, IGF-2

Definitions

  • the present invention relates to materials and methods involved in cartilage repair. Particularly, but not exclusively, it relates to the inhibition of apoptosis at the site of cartilage injury.
  • the present inventors have identified two major problems which exist in relation to cartilage repair. Firstly, there is a lack of integration of graft/repair tissue with the edge of the host defect, and secondly, the long-term survival of the repair tissue is generally poor, leading to eventual tissue breakdown.
  • the present inventors have studied the response of embryonic chick sternal cartilage and immature/mature bovine articular cartilage to wounding in vi tro . Interestingly, there is a remarkable similarity between the responses of embryonic and mature cartilage to wounding.
  • the present inventors have found that the initial response involves programmed cell death, or apoptosis, at the lesion edge (extending between 80-200 ⁇ m) and is subsequently followed by elevated proliferation behind the apoptotic lesion or renewed proliferation as in the case of mature tissue.
  • programmed cell death, or apoptosis at the lesion edge (extending between 80-200 ⁇ m) and is subsequently followed by elevated proliferation behind the apoptotic lesion or renewed proliferation as in the case of mature tissue.
  • chondrocytes within the apoptotic lesion die and those which do not, invariably divide .
  • the present inventors have found that if they replace the excised tissue when making a wound, apoptosis is markedly reduced. Furthermore, injecting the wound with agarose which sets within the lesion also has an inhibitory effect on cell death suggesting that if diffusible factors are inhibited or slowed from leaving the wound edge a reduction in apoptosis can be achieved.
  • the present invention provides a method for stimulating intrinsic cartilage repair comprising the step of inhibiting the apoptotic response to injury.
  • the apoptotic response to injury may be inhibited by the action of survival factors or by the action of a material which contacts the injury site thereby inhibiting the diffusible factors from leaving said injury site. Further, both actions may be used in combination.
  • a medicament for stimulating intrinsic cartilage repair comprising one or more survival factors capable of inhibiting an apoptotic response to injury of said cartilage .
  • the medicament further comprises a pharmaceutically acceptable carrier.
  • the present invention provides a method of treating cartilage injury in a patient, the method comprising administering a therapeutically effective amount of a survival factor to the patient.
  • the present invention provides a pharmaceutical composition comprising a survival factor in combination with a pharmaceutical carrier.
  • the present invention provides a medicament for inhibiting an apoptotic response to injury of cartilage comprising a material which when in contact with the cartilage injury site prevents diffusible factors from leaving said site.
  • a material for inhibiting an apoptotic response to cartilage injury forms a further aspect of the present invention.
  • the material is preferably a pharmaceutically acceptable material, such as medical plastics or agarose.
  • the material has the ability to change from a liquid to sold state to aid in its application to the injury site.
  • the term 'injury' may be taken to include any form of change in the cartilage from the normal condition. For example, changes after traumatic insult and/or degenerative changes in both osteo- and rheumatoid - arthritis.
  • survival factor is taken to include any agents that has the ability to inhibit apoptosis, i.e. promote the survival of a cell.
  • Preferred survival factors include growth factors that are known mitogens, for example, Insulin-like growth factor (IGF) I and II and Platelet-derived growth factor (PDGF) .
  • IGFs are particularly preferred due to their capabilities of rescuing fibroblasts (a connective tissue lineage cell) from proto-oncogene c-myc induced apoptosis (Harrington, 1994) .
  • BMPs bone morphogenetic proteins
  • BMPs bone morphogenetic proteins
  • Survival factors may also include agents used to prevent expression of genes associated with apoptosis. For example, disruption or down-regulation of proto-oncogene c- myc expression may inhibit the apopotic event. Such down regulation may be achieved by standard methods such as antisense mRNA, or disruption of associated promotor regions. Other proto-oncogenes known to suppress apoptosis may also be used, for example, Bcl -2.
  • the aspects of the present invention may further comprise a promoting factor or the use of a promoting factor for optimising the reparative response of the intrinsic cartilage.
  • These factors stimulate the rescued cells to participate in the reparative response. In other words, they may enhance the synthetic potential and proliferation of the rescued and surrounding chondrocyte cells in order to optimise the reparative response.
  • These promoting factors may include mitogenic competence factors, e.g. basic fibroblast growth factor, (bFGF) and cell cycle progression factors such as IGFs (which co-act as synthetic stimulators) and epidermal growth factors (EGF) .
  • bFGF basic fibroblast growth factor
  • IGFs which co-act as synthetic stimulators
  • EGF epidermal growth factors
  • Each of these factors may be administered in their natural form or in a recombinant form.
  • the promoting factors may be used in combination with BMP 2, 4, or 7 and/or GDF-5 recombinant proteins .
  • the present invention provides a method of screening for survival factors capable of inhibiting the apoptotic response to cartilage injury, the method comprising
  • survival factors may be formulated in pharmaceutical compositions.
  • These and/or promoting factors compositions may comprise, in addition, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
  • the precise nature of the carrier or other material may depend on the route of administration. The most preferred route of administration is for the carrier to be applied topically at the site of injury. However, other routes of administration will be known to the skilled person.
  • the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection.
  • Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.
  • the carrier may comprise a material, such as medically acceptable plastics, e.g. biopolymers, collagen gel or agarose, which may be injected directly into the wound site in cartilage.
  • the material for example agarose, may be loaded with one or more survival factors such as IGF I, PDGF, a variety of BMP's.
  • anabolic growth factors may also be included in the agarose, for example, TGF/3 or FGFs . All of these factors may in applied singly or in combination.
  • the factors are preferably mixed at low temperature setting agarose in the molten state (41°C) and injected directly into the wound site in cartilage. Once cooled the agarose will set in the injury site and the factors will diffuse out into the surrounding cells .
  • the pharmaceutically useful compound according to the present invention that is to be given to an individual, administration is preferably in a "prophylactically effective amount” or a “therapeutally effective amount” (as the case may be, although prophylaxis may be considered therapy) , this being sufficient to show benefit to the individual.
  • the actual amount administered, and rate and time-course of administration will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed) , 1980.
  • a pharmaceutical composition may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.
  • the survival factor and/or the promoting factors will be in form of a cream or ointment that may be applied cutaneously.
  • the cream or ointment preferably contains a diffusing agent that allows the factors to penetrate subcutaneous and diffuse to the site of injury.
  • Fig. 1 Seven-day bovine articular cartilage after wounding at day 0 (A,B), day 1 (C,D), day 5 (E,F), AND DAY 20 (G,H) .
  • Phase contrast images A,C,E,G
  • corresponding fluorescent TUNEL images B,D,F,H
  • Scale bar 100 ⁇ m.
  • Fig. 2. Tunel analysis after 5 days post-wounding. A) Apoptosis next to lesion edge (arrowed) ; b) when the lesion I filled with alginate, a marked reduction in apoptosis is observed. Fig. 3 and 4. Ultrastructural appearance of normal
  • the present inventors have employed two model systems to study the cell and molecular responses of cartilage to wounding .
  • the embryonic chick sternum has a number of advantages. It is a hyaline cartilage (unlike chick articular cartilage which is partly fibrous, Craig et al . , 1987) .
  • the sternum comprises two distinct chondrocyte sub-populations; the anterior portion which undergoes hypertrophy and subsequently ossifies and the posterior portion which is a permanent cartilage.
  • the sternum has similarities to articular cartilage but has the advantage that the permanent cartilages and the calcifying cartilage can be separated.
  • the perichondrium of the sternum is easily removed in entirety making it more akin to articular cartilage.
  • chick sterna are easily maintained in culture, indeed, increasing their size by some 4-fold over 10 days.
  • Embryonic chick sterna Sterna from day 17 embryos were removed aseptically, and the adhering muscle removed with the aid of watch-makers forceps. The perichondrium was removed in entirety. A wound @ 400 ⁇ m was made through the permanent cartilage of the posterior sternal keel with a seeker. The sterna were then placed in submerged organ culture and maintained in medium comprising Hams F12/DMEM (1:1), 10% foetal calf serum (FCS) , 1.4mM L-glutamine, ascorbic acid (50 ⁇ gml '1 ) and 1% antibiotic/antimycotic . Medium was changed every 2 days and cultures were maintained for up to 14 days. At time intervals from wounding to 14 days, specimens were harvested for a variety of analyses .
  • Apoptosis Using nick end labelling (TUNEL; and confirmed using fluorescently labelled Annexin V) the inventors were able to detect fluorescently labelled nuclei within 10 mins . of wounding around the immediate lesion edge . This pattern expanded to comprise a band of apoptosis ranging from 80-100 ⁇ m after two days in vi tro . The apoptotic nature of the chondrocyte death was confirmed at the ultrastructural level showing classic signs of programmed cell death including chromatin condensation and cell shrinkage (Fig.3 and 4) . After 7 days, detection of apoptotic nuclei by TUNEL declined leaving acellular areas around the lesion. After 14 days in vi tro, there was little evidence of repair.
  • Bovine articular cartilage One cm 2 full depth cartilage explants were taken from the metacarpal- phalangeal joints of 7 day cattle. Subsequently, 500 ⁇ m full depth lesions were made with a trephine and the explants cultured for up to 21 days in media identical to that described above.
  • the present inventors conducted a similar TUNEL analysis as that described for embryonic cartilage.
  • the response of articular cartilage to wounding was the same differing only in time-scale. Accordingly, the first fluorescently labelled nuclei appeared by 8 hours post-wounding. The first apoptotic chondrocytes were observed next to the lesion but subsequently spread inwards to occupy a band @ 100 ⁇ m by 24 hours and 150 ⁇ m within 5 days (Fig. 1) . This pattern of TUNEL labelling did not change significantly over the next 4 days.
  • the present inventors replaced the core of tissue back into the wound site immediately after removal with the trephine .
  • the majority of the apoptotic response was abolished except in regions where there was a significant gap (100 ⁇ m.>) between the tissue interfaces.
  • there was a graded response such that where the two tissue interfaces were closely opposed and there was physical contact, there was little or no apoptosis.
  • an apoptotic response could be observed which increased with gap size.
  • Cartilage explants (1cm 2 ) may be excised from metacarpo/metatarsophalangeal joints steers which are skeletally mature. Wounds (1.7mm diam.) are made in the centre of the explant with a trephine and maintained in serum-free (Hams F12/DMEM, 1:1) submerged culture.
  • explants can be harvested at 1, 2, 4, 8, 12, 16 and 24 and thereafter at 48 and 72 hr .
  • the expression of factors can then analysed at the gene and protein level.
  • RT-PCR quantitative reverse transcription polymerase chain reaction
  • si tu hybridization can be carried out using riboprobes .
  • Probes may be made by cloning cDNAs derived from RT-PCR into Bluescript vectors and, using the two RNA polymerase promotors, the sense and antisense probes can be generated.
  • riboprobes are either labelled with digoxigenin and detected by immunohistochemical techniques on thin sections or are labelled fluorescently so that thicker sections can be viewed in the confocal microscope which enables 3D reconstruction of the tissue architecture.
  • PCR primers can be designed covering regions of the gene that exhibit strong homology across a number of species. PCR products may be sequenced to confirm their identity.
  • the distribution of the expressed protein is ascertained by immunocytochemistry of protein either by indirect immunofluorescence or silver enhancement of colloidal gold conjugated secondary antibodies (Archer et al (1996) J. Anat. 189, 23-35; Morrison et al (1996) j. Anat . 189, 9-22). Both wax-embedded and cryo-processed material may be used as appropriate for the epitopes in question. Sections can be viewed by conventional light and fluorescent microscopy and scanning confocal microscopy. Specificity of the immunocytochemistry is confirmed by Western blotting. Experience with other projects utilising bovine material have found good species cross -reactivity with most antibodies.
  • the present applicants wished to investigate the relationship between the band of cell death which occurs at the lesion edge and the proliferation which occurs immediately behind it.
  • wounding stimulates a proliferative response but, for reasons unknown, those cells at the lesion edge enter the cell cycle but apoptose. Consequently, the present inventors probe a time course of wounded explant sections for specific markers of Gl entry such as cyclin D1/D3 and cdk4 to determine if chondrocytes near the lesion edge do enter the cell cycle prior to apoptosis which may have important implications for the experiments on the rescue of these cells (i.e. if the cells are rescued, they will go on to proliferate and contribute to the repair) .
  • Other experiments may include the probing for other cell cycle phase cyclins to determine at what phase the cells enter apoptosis .
  • C-myc expression can also activate other signalling pathways involving Ras which can suppress or promote apoptosis depending on downstream effectors (Kauffmann-Zeh et al (1997) Nature, 385, 544-548) . Such pathways may be important in the apoptosis/proliferative response the present inventors have seen in wounded cartilage. It is also known that oxidative products can stimulate chondrocytes (Ishizaki Y. , et al (1994) J. Cell Biol . , 126, 1069-1077) . And that death can be prevented by the presence of anti-oxidants such as cysteine .
  • the present inventors have shown that a number of growth factors (both BMPs and Growth and Differentiation factors, GDFs) are up regulated in the vicinity of a wound within hours.
  • This allows the mapping of the spatio- temporal pattern of factors known to regulate cartilage metabolism such as TGF3, IGF I and II, and BMPs 2, 4, 5, 6 and 9.
  • Such an analysis is useful as it can indicate an early stage modulation in phenotype.
  • BMP 6 is specific for hypertrophic chondrocytes whilst up-regulation of BMP 2 may indicate a switch to a more immature state.
  • BMPs can induce other factors such as FGFs which have been reported to promote cartilage repair (Cuevas P., et al (1988) Biochem.
  • cytokines IL-1 and TNF ⁇ can be added to the explant systems and the effects on the early repair responses can be analysed and compared and contrasted with non-cytokine added controls.
  • the data can provide important information as to the possible modulatory effects these cytokines may have on early response gene expression which in turn may have significant downstream effects.
  • Secondary response and attempted repair The above data can also be correlated with the downstream reparative responses on matrix synthesis (collagen, proteoglycan and glycoprotein expression including changes in integrin expression) .
  • agarose can be loaded with known survival factors such as IGF1, PDGF, a variety of BMPs together with anabolic growth factors such as TGF ⁇ and FGFs . These factors can be applied singly and in combination and the effects on gene expression and ultimately apoptosis analysed. Factors are mixed with low temperature setting agarose in the molten state (41°C) and injected directly into the wound site in cartilage held over ice thus setting the agarose. The data indicates factors that can be used therapeutically to inhibit apoptosis and undoubtedly enhance tissue integration during repair .

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Abstract

L'invention concerne l'utilisation d'un agent (tel qu'un facteur de survie ou de croissance) inhibant la réaction apoptotique à une lésion du cartilage et, éventuellement, un second agent (tel qu'un facteur de compétence mitogène, un facteur de développement de cycle cellulaire ou un facteur de croissance épidermique) favorisant la réaction de réparation du cartilage dans la préparation d'un médicament destiné à stimuler la réparation du cartilage.
PCT/GB1998/003089 1997-10-14 1998-10-14 Materiaux et methodes relatives a la reparation du cartilage WO1999018991A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU94512/98A AU9451298A (en) 1997-10-14 1998-10-14 Materials and methods relating to cartilage repair

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GBGB9721797.0A GB9721797D0 (en) 1997-10-14 1997-10-14 Materials and methods relating to cartilage repair
GB9721797.0 1997-10-14

Publications (1)

Publication Number Publication Date
WO1999018991A1 true WO1999018991A1 (fr) 1999-04-22

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PCT/GB1998/003089 WO1999018991A1 (fr) 1997-10-14 1998-10-14 Materiaux et methodes relatives a la reparation du cartilage

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AU (1) AU9451298A (fr)
GB (1) GB9721797D0 (fr)
WO (1) WO1999018991A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010007353A1 (fr) * 2008-07-18 2010-01-21 Smith & Nephew Plc Agents cataboliques
US20220143274A1 (en) * 2019-03-04 2022-05-12 Hans Ulrich Baer Biodegradable Mesh Implant for Soft Tissue Repair

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5368858A (en) * 1991-01-31 1994-11-29 Robert F. Shaw Methods and compositions for the treatment and repair of defects or lesions in cartilage
WO1997035607A1 (fr) * 1996-03-26 1997-10-02 Genetics Institute, Inc. Procedes d'induction tissulaire utilisant une combinaison de proteine morphogenetique osseuse et d'un peptide apparente a l'hormone parathyroidienne

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5368858A (en) * 1991-01-31 1994-11-29 Robert F. Shaw Methods and compositions for the treatment and repair of defects or lesions in cartilage
WO1997035607A1 (fr) * 1996-03-26 1997-10-02 Genetics Institute, Inc. Procedes d'induction tissulaire utilisant une combinaison de proteine morphogenetique osseuse et d'un peptide apparente a l'hormone parathyroidienne

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DUPREZ D ET AL: "Overexpression of BMP -2 and BMP -4 alters the size and shape of developing skeletal elements in the chick limb.", MECHANISMS OF DEVELOPMENT, (1996 JUL) 57 (2) 145-57, XP002092482 *
HARVEY A K ET AL: "Differential modulation of degradative and repair responses of interleukin-1-treated chondrocytes by platelet-derived growth factor.", BIOCHEMICAL JOURNAL, (1993 MAY 15) 292 ( PT 1) 129-36, XP002092480 *
TRIPPEL S B: "Growth factor actions on articular cartilage.", JOURNAL OF RHEUMATOLOGY. SUPPLEMENT, (1995 FEB) 43 129-32, XP002092481 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010007353A1 (fr) * 2008-07-18 2010-01-21 Smith & Nephew Plc Agents cataboliques
US20220143274A1 (en) * 2019-03-04 2022-05-12 Hans Ulrich Baer Biodegradable Mesh Implant for Soft Tissue Repair

Also Published As

Publication number Publication date
GB9721797D0 (en) 1997-12-17
AU9451298A (en) 1999-05-03

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