WO1999016406A2

WO1999016406A2 - Therapeutic method

Info

Publication number: WO1999016406A2
Application number: PCT/NZ1998/000145
Authority: WO
Inventors: Ian Reginald Reid; Jillian Cornish
Original assignee: Auckland Uniservices Ltd
Current assignee: Auckland Uniservices Ltd
Priority date: 1997-09-26
Filing date: 1998-09-25
Publication date: 1999-04-08
Anticipated expiration: 2000-03-26
Also published as: US6869926B1; EP1027027A4; JP2001524454A; US7208469B2; ATE348589T1; EP1027027B1; DE69836689D1; US20050171007A1; AU9369098A; EP1027027A2; WO1999016406A3

Abstract

This invention is directed to new therapeutic uses which involve the stimulation of chondrocyte proliferation. More particularly, it is directed to the use of amylin and adrenomedullin as agents which stimulate chondrocyte proliferation and which therefore have utility in the treatment of cartilage disorders and/or cartilage mediated bone growth.

Description

THERAPEUTIC METHOD

This invention is directed to new therapeutic uses which involve the stimulation of chondrocyte proliferation. More particularly, it is directed to the use of amylin and adrenomedullin as agents which stimulate chondrocyte proliferation and which therefore have utility in the treatment of cartilage disorders and/ or cartilage mediated bone growth.

BACKGROUND

Amylin is a 37-amino acid peptide cosecreted with insulin from the beta cells of the pancreatic islets. It was first reported by Cooper et al in Proceedings of the National Academy of Sciences, USA 84, 8628 (1987) and is the subject of European Patent 289287. Amylin has the following peptide sequence:

Lys-Cys-Asn-Thr-Ala-Thr-Cys-Ala-Thr-Gln- 1 5 10

Arg- Leu-Ala- Asn-Phe-Leu-Val- His-Ser-Ser- 11 15 20

Asn-Asn-Phe-Gly-Ala-Ile-Leu-Ser-Ser-Thr- 21 25 20

Asn-Val-Gly-Ser-Asn-Thr-Tyr

31 35

The native molecule contains a disulphide bridge between the cysteine residues shown at positions 2 and 7 in the primary structure, is amidated at its COOH- terminus, and is formed as a propeptide.

European Patent 289287 reports a number of biological effects including enhancement of hepatic glucose output, increased production of lactate from skeletal muscle and reduced action of insulin in skeletal muscle. Amylin is also reported in European Patent 408284 as having value for treatment of bone disorders and calcium imbalance. The patent specification attributes the activity of amylin to an inhibition of osteoclast motility. It is also reported in WO 96/02269 as stimulating bone growth through stimulating osteoblast proliferation.

Adrenomedullin is a 52-amino acid peptide first described in 1993 by Kitamura et al (Kitamura, K., et al. Adrenomedullin, a novel hypotensive peptide isolated from human pheochromocytoma. Biochem. Biophys. Res. Comτnun. 192:553-560 (1993)). It is present in normal adrenal/ medulla and in many other tissues including the atria, ventricles, endothelial cells, lungs, brain, kidneys and bone.

Adrenomedullin shows approximately 20% sequence identity with amylin and can therefore be termed a related peptide (Muff, R., et al. Calcitonin, calcitonin gene- related peptide, adrenomedullin and amylin: homologous peptides, separate receptors and overlapping biological actions. Eur. J. Endocrinol. 133: 17-20 (1995)). Both peptides have an NH₂ terminal ring created by a disulphide bond and are amidated at their COOH terminals.

Like amylin, adrenomedullin is also reported to have a range of activities. It is a potent vasodilator. It also has value in the treatment of bone disorders. This is primarily through an ability to stimulate osetoblast activity and proliferation in vitro and in vivo (Cornish, J., et al. Adrenomedullin is a potent stimulator of osteoblastic activity in vitro and in vivo. Am. J. Physiol (Endocrinol Metab) 36:E1113-E1120, (1997)).

However, to date, there has been no report of either of the peptides amylin or adrenomedullin, as having any effect on chondrocytes. It is the applicants finding that both of these peptides are effective in the stimulation of chondrocyte proliferation and therefore on the growth of both cartilage and lineal bone. This effect is believed to be mediated through a single receptor on chrondrocytes which underlies the applicant's invention. SUMMARY OF THE INVENTION

The invention has a number of aspects. In a first aspect, the invention provides a method of treating a patient to stimulate chondrocyte proliferation in vivo which comprises the step of increasing the active concentration of amylin within said patient.

Another aspect provides a method of treating a patient to stimulate chondrocyte proliferation in vivo which comprises the step of administering to said patient amylin or an analog thereof in an amount effective to stimulate chondrocyte proliferation.

In another embodiment, the invention provides a method of treating a patient to stimulate chondrocyte proliferation in vivo which comprises the step of increasing the active concentration of adrenomedullin within said patient.

In a further embodiment, the invention provides a method of treating a patient to stimulate chondrocyte proliferation in vivo which comprises the step of administering to said patient adrenomedullin or an analog thereof in an amount effective to stimule chondrocyte proliferation.

In still a further aspect, the invention provides a method of treating a patient to stimulate chondrocyte proliferation in vivo which comprises the step of activating the receptor localised on chondrocytes of said patient to which amylin and/or adrenomedullin bind.

Most preferably, the method involves activation of the adrenomedullin receptor.

Conveniently, in each of the above methods the stimulation of chondrocyte proliferation is part of a method of treating a patient to stimulate cartilage growth and/ or repair or to stimulate bone growth.

The invention also provides a method of stimulating chondrocyte proliferation in vitro which comprises administering an amount of amylin, adrenomedullin or an analog of either amylin or adrenomedullin to said chondrocytes which is effective in inducing chondrocyte proliferation. Other aspects include:

the use of amylin or an analog thereof in the preparation of a medicament for effecting chondrocyte proliferation;

the use of adrenomedullin or an analog thereof in the preparation of a medicament for effecting chondrocyte proliferation;

the use of a ligand which binds to and activates the receptor to which amylin and/or adrenomedullin binds (preferably the adrenomedullin receptor) in the preparation of a medicament for effecting chondrocyte proliferation;

the use of an amylin agonist in the preparation of a medicament for effecting chondrocyte proliferation;

the use of an adrenomedullin agonist in the preparation of a medicament for effecting chondrocyte proliferation;

the use of amylin-(l-8) in the preparation of a medicament for effecting chondrocyte proliferation; and

the use of adrenomedullin-(27-52) in the preparation of a medicament for effecting chondrocyte proliferation.

DESCRIPTION OF THE DRAWINGS

The present invention is broadly as defined above. However, it wϋl be appreciated by those persons skilled in the art that it is not limited thereto and that it also includes embodiments which are more particularly described below and illustrated by the experimental data presented. This data includes the information shown in the accompanying drawings in which: Figure 1 shows the effects of daily systemic administration of amylin for 4 weeks on growth plate width in the tibiae of normal adult male mice, n = 20 in each group. *, significantly different from control, P = 0.0002;

Figure 2 shows the effects of daily systemic administration of amylin for 4 weeks on bone length in the tibiae of normal adult male mice, n = 20 in each group. *, significantly different from control, P = 0.004;

Figure 3 shows the effect of the amylin fragment (amylin (1-8)) on epiphyseal growth plate width; and

Figure 4 shows the effect of the adrenomedullin fragment (adm 27-52) on epiphyseal growth plate width.

DESCRIPTION OF THE INVENTION

As broadly defined above, the present invention relates primarily to methods for stimulating chondrocyte proliferation. The invention therefore has utility in any application where stimulation of chondrocyte proliferation or growth is viewed as desirable, including for example cartilage growth and bone growth.

The applicants have found that chondrocyte proliferation is able to be effected using a number of related approaches. A first such approach is through a focus upon amylin. The applicants have found that increasing the effective concentration of amylin within a patient able to interact with the patients chondrocytes has the effect of stimulating chondrocyte proliferation.

Amylin for use in accordance with this approach can be obtained from any convenient commercial source (such as Bachem California, Torrence, CA, USA). Alternatively, amylin can be synthesised, using the procedure as described by way of example in EP 408284.

The amylin used can be homologous or heterologous to the patient to be treated.

For example, amylin from humans and other mammals eg. rat, monkey, dog, cat, mouse, guinea pig, hamster, degus, rabbit and hare can be used. The structure of these various peptides is reported in Endocrine Reviews 1994, 15(2) 163 by Garth J S Cooper which is incorporated herein by reference.

Most conveniently, the effective concentration of amylin will be increased through direct administration using either amylin itself or an amylin pro-drug (a form which is cleaved within the body to release amylin). It is however not the applicants intention to exclude increasing amylin concentration through administration of either amylin agonists (substances which effect a direct increase in the production or activity of amylin within the body, or inhibitors of amylin antagonists (substances which bind amylin or otherwise prevent or reduce the action of amylin within the body. These latter compounds exert an indirect effect on effective amylin concentrations through the removal of an inhibitory mechanism.

Another possibility is administration of a replicable vehicle encoding amylin to the patient. Such a vehicle (which may be a modified cell line or virus which expresses amylin within the patient) could have application in increasing the concentration of amylin within the patient for a prolonged period.

It is also contemplated that amylin analogs can be employed in this invention. As used herein "analog" means a protein which is a variant of another protein through insertion, deletion or substitution of one or more amino acids but which retains at least substantial functional equivalency.

A protein is a functional equivalent of another protein for a specific function if the equivalent protein is immunologically cross-reactive with, and has at least substantially the same function as, the original protein. The equivalent can be, for example, a fragment of the protein, a fusion of the protein with another protein or carrier, or a fusion of a fragment with additional amino acids. For example, it is possible to substitute amino acid in a sequence with equivalent amino acids using conventional techniques. Groups of amino acids normally held to be equivalent are:

(a) Ala, Ser, Thr, Pro, Gly;

(b) Asn, Asp, Glu, Gin;

(c) His, Arg, Lys; (d) Met, Leu, He, Val; and (e) Phe, Tyr, Trp.

In the case of amylin, the preferred analogs are fragments of the protein. In particular, amylin (1-8) can be used (ie. a fragment consisting of amino acids 1 to 8 of the amylin sequence) .

Functional equivalency of analogs can also be readily screened for by reference to the abihty of the analog to both bind to and activate the appropriate receptor.

In addition to the above approach, which focuses upon amylin and its analogs, the invention provides a further approach to chondrocyte proliferation. This second approach has a focus upon adrenomeduUin. The applicants have found that, in an equivalent manner to amylin, increasing the effective concentration of adrenomeduUin within a patient able to interact with the chondrocytes in that patient stimulates chondrocyte proliferation.

For use in this approach, adrenomedullin can be obtained from any convenient commercial source or, as is the case with amylin, synthesised using techniques weU known in the art. Such techniques include those described hereinafter.

Again, it is most convenient that the effective concentration of adrenomedullin be increased through direct administration using either adrenomedullin itself or an adrenomeduUin pro-drug. However, adrenomedullin agonists or inhibitors of adrenomedullin antagonists are not excluded.

As with amylin, adrenomedullin can also be administered in the form of a repUcable vehicle encoding adrenomedullin to the patient for release of adrenomeduUin over a prolonged period.

AdrenomeduUin analogs can also be employed. For this purpose, the term "analog" has the equivalent meaning of that given above for amylin. In the case of adrenomeduUin, a particularly preferred analog is adrenomeduUin (27-52) (ie. a fragment consisting of amino acids 27-52 of the adrenomedullin sequence).

The invention stiU further provides a third approach to chondrocyte proliferation. This further approach focuses upon the receptors on chondrocytes to which amylin and adrenomedullin bind and upon effecting chondrocyte proliferation through use of any Ugand which both binds to and activates these receptors.

It will be appreciated that amylin, analogs of amylin, adrenomedullin and analogs of adrenomeduUin are aU Ugands which achieve this. Indeed, the use of these substances as active agents represents a preferred aspect of the invention. However, it should be appreciated that this approach has not restricted the use of amylin, adrenomedullin and their analogs but also extends to any Ugand which fulfils the functional requirement of both binding to and activating (stimulating) the amylin or adrenomedullin receptors. Such additional Ugands are, for example, beUeved to include peptides such as calcitonin gene related peptide (Muff, R., et al. Calcitonin, calcitonin gene-related peptide, adrenomedullin and amylin: homologous peptides, separate receptors and overlapping biological actions. Eur. J. Endocrinol. 133: 17-20 (1995)).

A specific feature of this approach is to employ Ugands which bind to and activate the adrenomeduUin receptor. This receptor was described in, for example, Kapas, S., et al. Cloning and expression of cDNA encoding a rat adrenomeduUin receptor. J. Biol. Chem. 270:25344-25347 (1995). It is further described in Montuenga, L. M., et al. Expression of adrenomedullin and its receptor during embryogenesis suggests autocrine or paracrine modes of action. Endocrinology 138:440-451 (1997)).

Additional stimulatory Ugands can therefore, for example, be identified by a screening protocol employing at least the Ugand binding domain of the adrenomeduUin receptor. This screening method can, for example, utiUse the expression of the adrenomeduUin receptor in Xenopus oocytes using standard recombinant DNA methods and measurement of the adrenomeduUin receptor- mediated signal transduction evoked by novel stimulatory Ugands.

For therapeutic appUcation, the active compound (amylin, adrenomeduUin, analog or Ugand) will be formulated as a medicament. The details of the formulation will ultimately depend upon the active compound itself and upon the route of administration chosen. It will however be usual for the medicament to include combination of the active compound with a suitable carrier, vehicle or dUuent. Dosage rates wiU also be active compound and administration route dependent. However, by way of example, the dosage of active compound to be administered by injection wiU be in the range of 0.01- 100 mg/kg of body weight.

Further, whUe formulations in which the active compounds represent the sole active principle are most likely to be used, it is by no means intended that formulations which are suitable for combination therapies be excluded. The active compound can be administered together with any other therapeutic agent, including any other agent which has an effect on chondrocyte proliferation.

The invention, in its various aspects, will now be iUustrated by the experimental section which foUows. It will however be appreciated that the experiments are nonlimiting.

EXPERIMENTAL

METHODS

(a) Chondrocyte Monolayer Cell Cultures Fresh cartilage samples were coUected from the tibial plateaus and femoral condyles of mature, healthy crossbred dogs (2-4 years; 20-25 kg). The chondrocytes were isolated as previously described (Connective Tissue Research 1988; 18:205-222). Briefly, the chondrocytes were obtained by pronase and coUagenase digestion of the cartUage, then the ceUs were centrifuged, washed and resuspended in media before being cultured in 75 cm² tissue culture flasks. The ceUs were incubated under 5% CO2 and 95% air at 37°C. Confluence was reached by 7- 10 days, at which time the ceUs were subcultured. After trypsinization, the ceUs are rinsed and resuspended in fresh medium, then seeded at 5 x 10⁴ ceUs/ml in 24-weU plates (0.5 ml ceU suspension per weU, ie. 1.4 x 10⁴ ceUs/cm²). Proliferation studies (ceU counts and thymidine incorporation) were performed. Subconfluent population were changed to serum-free medium with 0.1% bovine serum albumin plus the experimental compounds. CeU numbers were analysed at 24 hours after addition of the peptide or vehicle. The ceU numbers were determined using a haemocytometer chamber. Results were expressed per weU. [³H]-thymidine incorporation was assessed by pulsing the ceUs with [³H]-thymidine (luCi/weU) two hours before the end of the experimental incubation. The experiment was terminated at 24 hours by washing the ceUs in media containing cold thymidine foUowed by the addition of 10% tricholoroacetic acid. The precipitate was washed twice with ethanol: ether (3: 1) and the weUs desiccated at room temperature. The residue was redissolved in 2 M KOH at 55°C for 30 mins, neutraUsed with 1 M HC1, and an aUquot counted for radioactivity. Results were expressed as dpm per weU. For both ceU counts and thymidine incorporation, each experiment was performed at least 4 different times using experimental groups consisting of at least 6 weUs.

(b) Chondrocytes 3-dimensional cell cultures in alginate beads Alginate head cultures were estabUshed as described by Guo, et al. Culture and growth characteristics of chondrocytes encapsulated in alginated beads. Connective Tissue Research. 19:277-297 (1989). Briefly the ceUs were suspended in a solution of 1.25% (wt/vol) alginate in HEPES (20 M HEPES buffer pH neutral) at a density of 2xl0⁶ ceUs/ml> The suspension of chondrocytes were slowly extruded through a 22-gauge needle in a dropwise manner into 40 ml of 0J M CaCl₂ solution. After instantaneous gelation, the beads were aUowed to further polymerise in CaCL2 solution (10 mins, room temperature). The beads were washed sequentiaUy, twice in 0J5 M NaCl and twice in Dulbecco's modified Eagle's medium (DME). After the washing procedure, the beads were placed into 24-weU culture plates (10 beads/weU) and fed with 1ml 10% fetal calf serum (FCS) SMW with 5μg/ml ascorbic acid. The cultures were maintained at 37°C in a humidified atmosphere of 5% C0₂ in air. The medium was changed every second day. On day 4 and 6 of culture, peptide or vehicle was added. CeU numbers were analysed at day 8 by exposing alginate beads to 50 mM ethylenediaminetetraacetic acid (EDTA) for approximately 10 minutes at 37°C. Counting was performed in a haemocytometer chamber. Results were expressed per weU. Tritiated-thymidine incorporation (³H-thymidine) was assessed by pulsing the beads with ³H-thymidine (lμCi/weU) 48 hours before the end of experimental incubation. Experiments were then terminated at day 8 of culture by dissolving the beads in 50 mM EDTA. The ceUs were washed twice with distilled water by centrifuging. PeUets were resuspended and counted for radioactivity.

(c) In Vivo Study: Experimental Design

Two groups of 20 sexuaUy mature male Swiss mice aged between 40 and 50 days and weighing 25-32g, were given daily subcutaneous injections (50 ul) in the loose skin at the nape of the neck for 5 days/week over 4 consecutive weeks. The treated group was injected with peptide at a dose of 300 ug/ kg/ injection and the control group was injected with vehicle (water). Animals were housed in a room maintained at 20°C on 12-hour Ught/dark cycles. They were fed diet 86 rodent peUets (New Zealand Stockfeed Ltd) ad Ubitum throughout the experiment. Each animal's weight was recorded at the beginning and end of the experiment. One day after the last injection, animals were sacrificed by cervical dislocation. They study had the approval of the local institutional review board.

The tibiae were dissected free of adherent tissue. Tibial lengths were recorded by measuring the distance between the proximal epiphysis and the distal tibio-fibular junction using an electronic micrometer (Digimatic CaUpers, Mitutoyo, Japan). Bones were placed in 10% phosphate-buffered formalin for 24 hours and then dehydrated in a graded series of ethanol solutions and embedded, undecalcified, in methylmethacrylate resin. Tibiae were sectioned longitudinaUy through the frontal plane and calvariae were cut cross-sectionaUy at the base of the parietal bone. AU sections were 4 um think and were cut on a Leitz microtome using a tungsten- carbide knife (Microknife Sharpening, Utah, USA). Sections were mounted on gelatin-coated sUdes and air-dried. They were stained with Goldner's tri-chrome and examined using an Olympus BX 50 microscope (Olympus Optical Co Ltd, Tokyo, Japan) which was attached to an Osteomeasure Image Analyzer (Osteometrics Inc. Atlanta, GA).

Tibial histomorphometric analyses were made from three adjacent sections one third of the way through the anterior/posterior depth of the proximal tibiae. Epiphyseal growth plate thickness was measured at three sites evenly spaced along its length. AU measurements were made by one operator who was blinded to the treatment group of each bone.

Materials Rat amylin was sourced from Bachem California, Torrance, CA, USA. LyophUised material was dissolved in water prior to administration. Methylmethacrylate was purchased from Acros Organics N.V., Geel, Belgium.

Rat amyUn-(l-8) used in this study was a COOH-terminal amide synthesized on methylbenzhydrylamine resin by standard soUd-phase techniques foUowed by hydrogen fluoride deprotection and cleavage from the resin. Amylin-(l-8) was cyclized in a dUute solution of 90% acetic acid by treatment with methanol solutions of iodine and purified to >96% homogeneity by reverse-phase high performance Uquid chromatograph (RP HPLC). Structures were confirmed by matrix- assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF system, model G2025 A, Hewlett Packard CA, USA) and amino acid analysis of acid hydrolysates %%4929%%.

Human adrenomeduUin and its fragments were synthesized on methylbenzhydrylamine resin using standard soUd-phase procedures, and cleaved with hydrogen fluoride /anisole. Sequences containing a disulfide bridge were cyclized by titration with h in 90% acetic acid/water solutions. Crude materials were purified by gel filtration on Sephadex columns in 50% acetic acid foUowed by gradient elution on C 18 siUca using acetonitrile/0J% trifluoroacetic acid eluants. Homogeneity of final peptides was assessed by thin layer chromatography, analytical HPLC, amino acid analysis and matrix-assisted laser-desorption- ionization mass spectroscopy. Purities were usuaUy >98%.

Statistical Analysis

Data are presented as mean ± sem. Where parameters have been measured more than once in each animal these values have been averaged to produce a single value for each animal before further analysis. The significant of treatment effects was evaluated using Student's t tests for unpaired data. These comparisons were specified a priori, so adjustment of α(0.05) was not necessary.

RESULTS

Amylin

(a) Chondrocyte Cell Cultures

Amylin influenced chondrocyte proliferation, increasing ceU numbers from 4J2 ± 0.23 (xlO⁴) (mean ± sem) in control ceUs to 5.1 1 ± 0.21 (xlO⁴) in those ceUs incubated with amylin (p=0.01) as weU as increasing thymidine incorporation (ie. DNA synthesis) from 20725 ± 997 dpm in control ceUs to 25937 ± 1203 dpm in amylin-treated ceUs. (b) Chondrocytes 3-dimensional cell cultures in alginate beads

Amylin again influenced chondrocyte proliferation, increasing ceU numbers from 5.58 ± 0J6 (xlO⁴) (mean ± sem) in control ceUs to 6.07 ± 0.05 (xlO⁴) in those ceUs incubated with amylin (10 ¹⁰M) (p<0.03) as weU as increasing thymidine incorporation (ie. DNA synthesis) from 1 135 ± 85 dpm in control ceUs to 2584 ±229 dpm in amylin-treated ceUs (p<0.001).

(c) In Vivo Study

Amylin influenced the tibial growth plate, increasing its width from 0.083 ± 0.005 mm (mean ± sem) in the control animals to 0J08 ± 0.003 mm in those receiving amylin (P = 0.0002) (Figure 1). The total length of the tibiae was also increased from 11.31 ± 0.07 mm in control animals to 11.67 ± 0.09 mm in animals injected with amylin (P-0.004) (Figure 2).

Amylin 1-8

(a) Amylin- (1-8) also influenced chondrocyte proliferation, increasing ceU numbers from 3.23 ± 0.11 (xlO⁴) (mean ± sem) in control ceUs to 3.63 ± 0.09 (xlO⁴) in those ceUs incubated with amyUn-(l-8) (10-8M) (p = 0.02) as weU as increasing thymidine incorporation (DNA synthesis) from 26859 ± 423 dpm in control ceUs to 28932 ±628 dpm in amylin=(l-8) treated ceUs (p = 0.02).

(c) The growth plate width in the proximal tibiae of mice injected systemicaUy with amylin-(l-8) is significantly increased compared to control animals (mean ± sem: 0J 11 mm ± 0.004 compared to 0.081 mm ± 0.004; p<0.0001). See Figure 3.

Adrenomedullin

(a) AdrenomeduUin influenced chondrocyte proliferation, increasing ceU numbers from 1.79 ± 0.07 (x 10⁴) (mean ± sem) in control ceUs to 2.27 ± 0J2 (x 10⁴) in those ceUs incubated with adenomedullin (10 M) (p<0.01).

Adrenomedullin-(27-52)

(c) AdrenomeduUin- (27- 52) increased the growth plate width from 0.094 mm ± 0.003 (mean ± sem) in control animals to 0J 1 mm ± 0.003 in adrenomedullin- (27- 52) (p=0.003). See Figure 4. INDUSTRIAL APPLICATION

The above results clearly show that amylin and its anlogs (amylin- (1-8), for example) has the abiUty to stimulate chondrocyte proliferation. Simflarly, adrenomeduUin and its analogs (adrenomeduUin-(27-52) have equivalent capability.

The results additionaUy show the ability of both amylin, adrenomeduUin and their analogs to influence the growth of cartUage as weU as increased bone growth. This latter effect is consistent with the formation of bone on a template of cartUage tissue.

Both amylin and adrenomeduUin are beUeved to be exerting the effect on chondrocyte proliferation/ cartUage growth/bone formation through the mediation of the amylin /adrenomedullin receptor.

The present invention therefore provides new approaches to chondrocyte proliferation. These involve firstly increasing the active concentration of amylin/adrenomedullin in a patient and secondly the activation of the amylin/adrenomeduUin receptor locaUsed on chondrocyte ceUs.

The approaches of the invention have appUcation in the treatment of patients in a variety of conditions. Principal amongst these are conditions where the patient is suffering from a cartUage defect, either through injury or through degenerative, inflammatory or other disease.

The approaches of the invention also have appUcation in the stimulation of bone growth, particularly lineal bone growth. This provides the invention with appUcation in treating patients (for example, chUdren) who are of short stature or who otherwise suffer from defects which would benefit from stimulation of the growth of limb bones.

The invention also has appUcation in vitro. Extracted chondrocytes can be proliferated using the present methods. The proliferated chondrocytes can then be employed in methods of therapy, particularly those which involve the treatment of damaged cartUage. It will be appreciated by those persons skilled in the art that the above description is provided by way of example only and that numerous changes and variations can be made whUe stiU being within the scope of the invention as defined by the appended claims.

Claims

1. A method of treating a patient to stimulate chondrocyte proliferation in vivo which comprises the step of increasing the active concentration of amylin within said patient.

2. A method of treating a patient to stimulate cartUage growth and/ or repair in vivo through stimulation of chondrocyte proliferation which comprises the step of increasing the active concentration of amylin within said patient.

3. A method of treating a patient to stimulate bone growth in vivo through stimulation of chondrocyte proliferation which comprises the step of increasing the active concentration of amylin within said patient.

4. A method according to any one of claims 1 to 3 wherein the active concentration of amylin is increased through administration of amylin to said patient.

5. A method according to any one of claims 1 to 3 wherein the active concentration of amylin is increased through administration of an amylin agonist.

6. A method of treating a patient to stimulate chondrocyte proliferation in vivo which comprises the step of administering to said patient amylin or an analog thereof in an amount effective to stimulate chondrocyte proliferation.

7. A method of treating a patient to stimulate cartUage growth and/or repair in vivo through stimulation of chondrocyte proliferation which comprises the step of administering to said patient amylin or an analog thereof in an amount effective to stimulate chondrocyte proliferation.

8. A method of treating a patient to stimulate bone growth in vivo through stimulation of chondrocyte proliferation which comprises the step of administering to said patient amylin or an analog thereof in an amount effective to stimulate chondrocyte proliferation.

9. A method according to any one of claims 6 to 8 wherein amylin is administered to said patient.

10. A method according to any one of claims 6 to 8 wherein an analog of amylin is administered to said patient.

11. A method according to claim 10 wherein said amylin analog is amylin- ( 1-8).

12. A method of treating a patient to stimulate chondrocyte proliferation in vitro which comprises the step of increasing the active concentration of adrenomeduUin within said patient.

13. A method of treating a patient to stimulate cartUage growth and/or repair in vivo through stimulation of chondrocyte proliferation which comprises the step of increasing the active concentration of adrenomeduUin within said patient.

14. A method of treating a patient to stimulate both growth vivo through stimulation of chondrocyte proliferation which comprises the step of increasing the active concentration of adrenomedullin within said patient.

15. A method according to any one of claims 12 to 14 wherein the active concentration of adrenomedullin is increased through administration of adrenomeduUin to said patient.

16. A method according to any one of claims 13 to 15 wherein the active concentration of adrenomeduUin is increased through administration of an adrenomeduUin agonist.

17. A method of treating a patient to stimulate chondrocyte proliferation in vivo which comprises the step of administering to said patient adrenomeduUin or an analog thereof in an amount effective to stimule chondrocyte proliferation.

18. A method of treating a patient to stimulate cartUage growth and/ or repair in vivo through stimulation of chondrocyte proliferation which comprises the step of administering to said patient adrenomeduUin or an analog thereof in an amount effective to stimulate chondrocyte proliferation.

19. A method of treating a patient to stimulate bone growth in vivo through stimulation of chondrocyte proUferation which comprises the step of administering to said patient adrenomeduUin or an analog thereof in an amount effective to stimulate chondrocyte proliferation.

20. A method according to any one of claims 17 to 19 wherein adrenomeduUin is administered to said patient.

21. A method according to any one of claims 17 to 19 wherein an analog of adrenomeduUin is administered to said patient.

22. A method according to any one of claims 17 to 19 wherein said adrenomedullin analog is adrenomeduUin- (27-52).

23. A method of treating a patient to stimulate chondrocyte proliferation in vivo which comprises the step of activating a receptor locaUsed on chondrocytes of said patient to which amylin and/ or adrenomeduUin binds.

24. A method of treating a patient to stimulate cartUage growth and/or repair in vivo through stimulation of chondrocyte proliferation which comprises the step of activating a receptor locaUsed on chondrocytes of said patient to which amylin and/ or adrenomedullin binds.

25. A method of treating a patient to stimulate bone growth in vivo through stimulation of chondrocyte proliferation which comprises the step of activating a receptor locaUsed on chondrocytes of said patient to which amylin and/ or adrenomeduUin binds.

26. A method according to any one of claims 23 to 25 wherein the receptor which is activated is the adrenomedullin (ADM) receptor.

27. A method according to any one of claims 23 to 26 wherein receptor activation is effected through administration of a Ugand which binds to and activates the receptor.

28. A method according to any one of claims 23 to 26 wherein receptor activation is effected through administration of amylin.

29. A method according to any one of claims 23 to 26 wherein receptor activation is effected through administration of an amylin analog.

30. A method according to claim 29 wherein the amylin analog is amylin-(l-8).

31. A method according to any one of claims 23 to 26 wherein ADM receptor activation is effected through administration of adrenomeduUin.

32. A method according to any one of claims 23 to 26 wherein receptor activation is effected through administration of an adrenomeduUin analog.

33. A method according to claim 32 wherein the adrenomeduUin analog is adrenomeduUin-(27-52).

34. A method of stimulating chondrocyte proliferation in vitro which comprises administering an amount of amylin, adrenomeduUin or an analog of either amylin or adrenomeduUin to said chondrocytes which is effective in inducing chondrocyte proliferation.

35. A method according to claim 34 wherein an effective amount of amylin is administered.

36. A method according to claim 34 wherein an effective amount of an amylin analog is administered.

37. A method according to claim 36 wherein the amylin analog is amylin- 1-8.

38. A method according to claim 34 wherein an effective amount of adrenomedullin is administered.

39. A method according to claim 34 wherein an effective amount of an adrenomeduUin analog is administered.

40. A method according to claim 39 wherein the adrenomeduUin analog is adrenomeduUin- 27-52.

41. The use of amylin or an analog thereof in the preparation of a medicament for effecting chondrocyte proliferation.

42. The use of adrenomeduUin or an analog thereof in the preparation of a medicament for effecting chondrocyte proliferation.

43. The use of a Ugand which binds to and activates a receptor locaUsed on chondrocytes to which amylin and/ or adrenomeduUin binds in the preparation of a medicament for effecting chondrocyte proliferation.

44. The use of claim 43 wherein the Ugand is one which binds to and activates the adrenomeduUin (ADM) receptor.

45. The use of any one of claims 41 to 44 wherein the medicament is for the growth and/ or repair of cartUage.

46. The use of any one of claims 41 to 44 wherein the medicament is for the growth of bone.

47. The use of claim 46 wherein the medicament is for effecting the lineal growth of bone.

48. The use of an amylin agonist in the preparation of a medicament for effecting chondrocyte proliferation.

49. The use of an adrenomeduUin agonist in the preparation of a medicament for effecting chondrocyte proliferation.

50. The use of amylin-(l-8) in the preparation of a medicament for effecting chondrocyte proliferation.

51. The use of adrenomeduUin-(27-52) in the preparation of a medicament for effecting chondrocyte proliferation.