GROWTH FACTOR-DEPENDENT DISEASES

Abstract: A method of identifying or developing a compound which is useful in treating a growth factor-dependent disease, the method comprising the step of determining whether the compound modulates the amount, function or structural integrity of a growth factor binding protein.

Description: GROWTH FACTOR-DEPENDENT DISEASES This invention relates to the provision of drugs for the treatment or prevention of growth factor-dependent diseases including cancer or osteoporosis. More particularly, based on new data, it provides an experimental approach to the identification of such drugs and their development. As is described in more detail below, growth factor- dependent diseases include any disease in which the phenotype may be modified by changes in the availability or function of growth factors or related molecules. The invention embraces an endpoint-based experimental approach to the identification of novel drug therapies for such diseases, with such endpoints being the amount, structural integrity, or function of growth factor-binding proteins (GFBPs) . In the context of the present invention, prostate cancer is a growth factor- dependent disease. Prostate cancer often metastasises to bone, where it typically causes a sclerotic (osteoblastic, 'bone-forming') response which distinguishes this disorder from most other (osteolytic, 'bone-destroying') causes of bone metastasis. Whereas osteolytic radiographic appearances could reflect bone destruction by tumor, the pathogenesis of osteoblastic disease remains unexplained. One class of molecules which could be involved in this phenomenon are the polypeptide growth factors, several of which enhance stromal osteoblast growth and/or function. These include transforming growth factor- β, platelet-derived growth factor-BB, basic fibroblast growth factor, and insulin-like growth factors (IGFs) I and II . Several of these growth factors are sequestered by growth factor binding proteins (GFBPs) present in either extracellular matrix (transforming growth factor-β, basic fibroblast growth factor, platelet-derived growth factor-BB) or serum (IGFs). with the main serum binding protein for IGF- I being insulin-like growth factor binding protein-3 (IGFBP-3). Changes in expression of these growth factors are inducible in prostate cancer cell lines following hormonal manipulation and other stimuli 1 ,2 . Nonetheless, the contribution of any one growth factor to the prostate cancer phenotype remains uncertain. Prostate-specific antigen (PSA) is a serine protease synthesised predominantly, though not exclusively, by prostatic epithelial cells. Detectable amounts are also measurable in the sera of normal males 3 , the breasts of normal females , and in association with other benign and malignant disorders such as breast cysts , breast cancers and lung cancer'. The clinical importance of PSA has been heretofore regarded exclusively in terms of its role as a prostate cancer diagnostic marker, either in the context of disseminated disease under treatment (most commonly), early diagnosis in screened populations, immuno- histochemical diagnosis in metastatic disease of unknown primary, investigation of symptomatic individuals with a past history of prostate cancer, or follow-up of asymptomatic individuals previously treated for primary prostate cancer . The physiological function of PSA involves cleavage of fibrous proteins in seminal vesicle secretions, thus leading to liquefaction of semen and enhancement of sperm motility 9. However, like other proteases linked with prostate cancer, such as urokinase " " and cathepsin D , PSA is reported to cleave numerous other proteins in vitro. These substrates include insulin, interleukin-2, fibrinogen, ovalbumin , parathyroid hormone-related protein 1' ", transforming growth factor-β 1 and IGFBP-3 17 . With respect to the latter substrate, in vitro data have raised the possibility of an autocrine-like enhancement of prostate cancer cell growth consequent upon proteolysis of IGFBP-3, associated with apparent potentiation of IGF-II activity 12 . As if to contradict this model, however, a similar study using the same cell line (PC-3) demonstrated significant enhancement of IGF-dependent growth by IGFBP-3 addition 18 . The latter result is consistent with a number of other studies suggesting mitogenic potentiation, rather than antagonism, between IGFs and IGFBP-3 19 . A possible explanation for this phenomenon is that IGFBP-3 blocks the down-regulation of the IGF-1 receptor (Conover & Powell (1991) Endocrinol. 129, 710-716). Whereas these latter two studies relate to a PSA-negative cell line, inverse correlations have been noted between serum IGFBP-3 and PSA levels in patients with metastatic prostate cancer "" . However, the latter study detected an even more striking increase in IGFBP-2 levels in such patients; this is a surprising finding given that PSA also cleaves IGFBP-2 in vitro 11 , and one which might lead one of ordinary skill in the art to surmise that the origin of the IGFBP-3 reduction in metastatic prostate cancer is unrelated to PSA action. There has been no evidence presented that PSA is involved in the metastatic prostate cancer phenotype and, based on the disclosures in the art, the association could well be indirect: PSA degrades insulin 14 , for example, which in turn normally maintains IGFBP-3 expression level 23 . Reference 25 shows that high levels of PSA are growth inhibitory to prostate cancer cells. Similarly, since androgens enhance both PSA 24,25 and EGF 26 expression within the prostate, and since EGF reduces cellular IGFBP-3 levels 27 , low IGFBP-3 levels could occur via systemic dysregulation of an androgen-dependent pathway independent of PSA. In addition, reduced plasma IGFBP-3 is also recognized in association with non-specific stresses such as those which occur postoperatively 2 " . Such stresses might plausibly be operative in metastatic cancer patients. Hence, low IGFBP-3 levels in metastatic prostate cancer cannot be assumed to imply PSA-dependent cleavage of the intact molecule on the basis of published findings. Expression of PSA within prostatic tissue has also been noted to coincide with low tissue levels of IGFBP-3 " . Two considerations cloud the interpretability of this finding, however. First, other authors have reported that high intraprostatic zinc levels fully inhibit PSA activation within the gland , implying that PSA acts only within the ejaculate and not within prostatic tissue itself, thereby preventing autodigestion of the gland. Second, no quantitative correlation was found in this study between PSA expression levels and reduced IGFBP-3 , thus bringing into question the causality of this association. Growth-inhibitory effects of IGFBPs have been noted in stromal cell systems , including osteoblasts ' , and also in prostate cancer cell lines " , consistent with induction of reduced IGF bioavailability. However, the IGFBP-3 gene has been shown to be a downstream target of the DNA damage-inducible wild-type, but not mutant, p53 gene " \ These and other observations raise the possibility that the growth-inliibitory effect of IGFBPs in cell culture may be related not to IGF binding but to membrane interactions which independently influence cell growth control . Indeed, in many cell systems IGFBP-3 expression potentiates, rather than antagonizes, the functional activity of IGFs 19 , implying a role for IGFBP-3 in reducing IGF receptor downregulation. These considerations caution against assuming a causal relationship between IGFBP proteolysis and tumor cell growth. Thus, previous work describing an in vivo interaction between PSA and IGFBP-3 and the IGFs is both inconclusive and inconsistent, and falls short of implicating either molecule as a therapeutic target. The only therapeutic target generally acknowledged in the clinical field of prostate cancer is the androgen receptor signalling pathway . Consistent with this, PSA is an androgen-inducible gene ; so too, however, are a number of known prostate cancer cell mitogens including epidermal growth factor and transforming growth factor- 1. The possibility that growth factor bioavailability may promote prostate cancer progression receives indirect support from clinical studies using the putative growth factor antagonist suramin 33 . However, in vitro studies indicate that suramin reduces prostate cancer cell expression of PSA, consistent with other reports suggesting that growth factors may mimic androgen action 36 , in the absence of associated growth inhibition " . Hence, no convincing in vivo evidence for the involvement of PSA in the pathogenesis or progression of prostate cancer is available. Recent reviews and editorials on the subject of PSA have been correspondingly silent on the possibility of its future role as a therapeutic drug target ' " . IGFs are implicated in the maintenance of bone structure and hence, potentially, could also be implicated in the pathogenesis of osteoporotic bone disease. Given that PSA overexpression is predominantly associated with prostate cancer (an exclusively male disease) whereas osteoporosis is mainly an affliction of postmenopausal women, it is perhaps unsurprising that little connection has been made within the literature between the osteoblastic phenotype of metastatic prostate cancer and novel therapeutic (including preventative) approaches to osteoporosis. The few passing comments on this subject in the literature have been overshadowed by extensive discussion as to the use of IGF- and/or growth hormone-based therapies 41 The present invention stems from the observation that bone density increases, both within prostatic bone metastases and possibly also in peripheral normal bone, in tandem with a selective reduction of intralesional IGFBP-3 levels. These increases quantitatively parallel tumor-dependent expression of the androgen-inducible IGFBP-3 protease PSA in metastatic lesions and peripheral blood respectively, but do not correlate with intralesional expression levels of osteoblast-stimulatory growth factors. This implies the existence of a paracrine-like growth loop dependent upon an IGFBP-3 protease within prostate cancer bone metastases, most likely PSA or other PSA-dependent proteases such as urokinase, and indicates that therapies which enhance this protease may be useful in the treatment of osteopaenic bone diseases such as osteoporosis. In vitro inhibition of androgen-dependent growth of LNCaP prostate cancer cells by addition of IGFBP-3 implies coexistence of an autocrine growth loop. These, and other observations of mine which are described in more detail below, indicate that the systematic identification of drugs for modulation of proteases which are capable of altering a function of a growth factor binding protein, where the growth factor bound by the protein is implicated in a disease state, provides a heretofore unrecognized therapeutic route for the treatment, amelioration and prevention of growth factor-dependent diseases. Thus, an object of the invention is to provide assays and screening methods in order to identify and develop new drugs suitable for treatment of such growth factor-dependent diseases. A further object of the invention is to provide new methods of treatment for growth factor-dependent diseases, particularly prostate cancer and osteopaenic bone diseases, such as osteoporosis, which are dependent on growth factors. The invention includes growth factor (GF) binding protein (GFBP)-based assays for the identification and development of candidate drugs for the treatment of growth factor-dependent diseases including (but not restricted to) prostate cancer and osteoporosis; and new uses for angiotensin- converting enzyme inhibitors (eg lisinopril, enalapril, captopril) in the treatment of prostate cancer and other insulin-like growth factor binding protein-3 (IGFBP3)-dependent diseases. I have provided clinical evidence substantiating a likely pathogenetic link between the expression of PSA, the occurrence of the osteoblastic phenotype, and the reduction of intralesional IGFBP3 concentrations; in vitro corroboration of the clinically-derived hypothesis as to a mechanistic link between the action of a GFBP protease and the in vivo behaviour of target cells of interest; detailed methods for the systematic identification and/or development of candidate drugs using GFBP-based assays; and an illustration of the feasibility of the approach using of the GFBP-based methodologic approaches, and the identification of a new antineoplastic use for certain known drugs. A first aspect of the invention provides a method of identifying or developing a compound which is useful in treating a growth factor- dependent disease, the method comprising the step of determining whether the compound modulates the amount, function or structural integrity of a growth factor binding protein. The growth factor binding protein is a binding protein associated with the growth-factor dependent disease, and compounds which modulate the amount, function or structural integrity of the GFBP may be useful in treating the disease. The compound may be one which increases or decreases the expression of the GFBP, thereby altering the amount, or it may be a compound which modifies the function of the GFBP, or it may be a compound which alters the structural integrity of the GFBP, for example it may lead to degradation of the GFBP. Modulation of the amount of GFBP may lead to consequential modulation of the amount of, or biological function of, a growth factor which binds to the GFBP. Thus, an increase in the amount of GFBP may lead to sequestration of growth factor thereby reducing the amount of biologically active growth factor; alternatively, depending on the growth factor, binding of the growth factor to the GFBP may lead to an increase in the biological activity of the growth factor since, in some circumstances, the growth factor may need to be presented to the growth factor receptor by a GFBP. A decrease in the amount of GFBP may, in some circumstances, lead to an increase in the amount of biologically active growth factor, but. as discussed above, since for certain growth factors binding to the GFBP may be required for binding or interaction of the growth factor with its receptor, a decrease in the amount of GFBP may lead to a decrease in growth factor-mediated activity. Modulation of the function of the GFBP by the compound may include, for example, alterations to the GFBP " s ability to bind a growth factor, or it may include, for example, alteration of the GFBP's ability to present the growth factor to the growth factor receptor, or it may, for example, include alteration of some other function of the GFBP, for example, a change in the kinetic constants with which it binds the growth factor or a change in the ability of the GFBP to bind one growth factor compared to its ability to bind another growth factor. Modulation of the strucmral integrity of the GFBP by the compound may involve any modulation of the strucmral integrity. For example, the compound may be one which leads to the degradation of at least part of the GFBP, or it may be one which confers resistance on the GFBP to degradation. As is described in more detail, the compound may act directly on the GFBP or it may act indirectly, for example by acting on a component which acts directly or indirectly on the GFBP. As is described below, and without limitation to the scope of the invention, components which act directly or indirectly on the GFBP, modulation of which may lead to modulation of the amount, function or strucmral integrity of the GFBP, include a growth factor which binds to the GFBP, a protease which acts on the GFBP, a natural inhibitor of such a protease and a further protease which acts on the GFBP protease. In one embodiment of the invention, the compound modulates the activity of a protease, which protease is capable of modulating the amount, function or strucmral integrity of the GFBP. In another embodiment of the invention, the compound modulates the binding of the growth factor to the GFBP. By "growth factor-dependent disease" we include the meaning that the disease is any in which the phenotype may be modified by changes in the availability or function of growth factors or related molecules. Such diseases include, but are not limited to, cancers and benign neoplastic disorders, premature senescence or dementia, osteopaenic bone disease including osteoporosis, baldness, acne, fibrotic disorders and diseases of abnormal angiogenesis. By "growth factor" we include any non-enzymatic secreted or membrane- bound or extracellular molecule which transduces signals and/or effects a cellular response by binding to a heterologous molecule at or near the cell surface. Thus, "growth factors" include polypeptide growth factors which are capable of binding transmembrane kinases (eg epidermal growth factor (EGF), transforming growth factor beta (TGFβ)); cytokines which are capable of binding hetero-oligomeric non-catalytic cytokine receptors (eg erythropoietin. growth hormone, interleukins and interferons) ; peptide hormones which are capable of binding seven-transmembrane-domain G- protein coupled receptors such as adrenalin, dopamine and serotonin; and neurotransmitters which are capable of binding ligand-gated ion channels such as acetylcholine and GABA. By "growth factor receptor" we include any transmembrane protein which binds a growth factor and, as an immediate and integral consequence, transduces an intracellular signal. By "growth factor binding protein" (GFBP) is included any membrane- attached or extracellular protein which binds a growth factor without directly transducing an intracellular signal as an immediate and integral consequence of such binding eg extracellular matrix proteoglycans, shed extracellular domains of transmembrane receptors. In addition, in the context of the invention, a growth factor binding protein includes a precursor form of certain growth factors which are produced as integral complexes of a growth factor and a binding protein thereto. Such growth factors include EGF, TGFα, hepatocyte growth factor and heregulin. It will be appreciated that, ill the context of the invention, the GFBP is typically the natural, cognate binding protein for a growth factor within the animal, and does not include proteins which accidentally bind the growth factor but are of no physiological significance. However, it will be appreciated that, because of the relatedness of certain growth factor molecules and growth factor binding proteins between animal species, a growth factor from one species may bind a growth factor binding protein from another related species. By "a protease which protease is capable of modulating the amount, function or strucmral integrity of a growth factor binding protein" is included a GFBP protease (or binding protein protease (BPP)) and any proteolytic enzyme which can cleave a given growth factor binding protein, irrespective of the functional consequence of such cleavage. The cleavage of the GFBP by the protease alters the GFBP such that it alters the biological effect of a particular growth factor, for example, it alters it such that the growth factor may be more or less mitogenic. In addition, the functional alteration of a GFBP may be with respect to a particular growth factor. For example, it is possible that inactivation may lead to reduction of binding of one growth factor but retention of binding activity towards another growth factor. Conveniently, the protease is a protease whose physiological role is to cleave the GFBP; thus, in particular preference the protease is selective for the GFBP. By "BPP protease" is included any proteolytic enzyme which can cleave a GFBP protease, irrespective of whether such cleavage activates, inactivates, or leaves unaffected the subsequent activity of the substrate BPP. Hence, a basic scheme of the interrelationship between these molecules is: BPP - BPP - GFBP - GF + GFR protease -> -> -> -» ( + )* ( + )* (* usually inhibitory but sometimes potentiating) It will be appreciated that the growth factor, or growth factor binding protein, or GFBP protease may be from any animal species. It is particularly preferred if these molecules are from a mammal such as man, rat, mouse, cow, horse, sheep, dog, cat or the like. It is most preferred if these molecules are of human origin, particularly when the methods or assays of the invention are used to identify compounds which are useful in treating a human disease. Growth factors as defined are well known in the art. Similarly, growth factor binding proteins and GFBP proteases are well known in the art. Nucleotide sequences of cDNAs encoding these proteins may readily be obtained from, for example, the GenBank or EMBL databases and suitable cDNAs for expression may be obtained by methods well known in the art. In any case, for any given growth factor, the cognate binding protein or proteins may be readily identified by the person skilled in the art. For example, the binding protein may be identified by using a radioligand binding assay (using for example, radio-iodinated growth factor) to detect the binding protein which binding protein can be identified by peptide sequencing. In carrying out the screening method of the invention, or assay as it may be termed, it is preferred if the disease is one involving known or suspected GFBP protease (BPP) dysregulation or one in which a GFBP may play a role. Typically, the protease is capable of degrading the growth factor binding protein by enzymatic proteolysis. It will be appreciated that the protease may act at one or more sites in the binding protein and that proteolysis typically leads to functional modification of the binding protein which in turn modifies the binding or in vivo function of the growth factor. The screening method or assay may be used as a primary screening method or assay in order to identify candidate compounds which may be therapeutically useful in their own right, or which may be useful as lead compounds to develop further. The screening method or assay may also be used in later stages of drug development, for example, in screening compounds synthesised based on the structure and property of the lead compounds. Conveniently, the screening method is carried out using a series of test compounds. The test compounds may be any suitable compound for screening. Conveniently, it will be a compound from a library of compounds which have been prepared, or are already available, for screening. The compound identified in the screen may be any compound which modulates the amount, function or strucmral integrity of a growth factor binding protein. As noted above, the compound may act directly or indirectly on the GFBP. in particular it may act on a component which leads to modulation of the amount, function or strucmral integrity of the GFBP. For example, the compound may be one which modulates the activity of a protease, which protease is capable of modulating the amount, function or structural integrity of a GFBP. The compound may act either directly or indirectly on the protease to modulate its activity. For example, the compound may be one which inhibits or stimulates the enzyme activity of the protease ("direct" effect), or it may be one which prevents or reduces or enhances the expression of the gene encoding the protease, or it may be one which modulates the availability of a natural inhibitor of the protease (such as a serine protease inhibitor or serpin) ("indirect effect"). The compound may be one which increases the activity of the said protease (compared to the activity in the absence of the test compound), or the compound may be one which decreases the activity of the said protease (compared to the activity in the absence of the test compound). It is preferred if the compound acts directly on the GFBP, for example by modulating binding of the growth factor to the GFBP, but the compound may act directly on another component such as the GFBP protease. It is particularly preferred if the compound is selected from a library of compounds which have been synthesised with a view to them being screened in an assay of the invention. For example, the library of compounds may have been synthesised with a view to them being protease inhibitors. Many libraries of potential protease inhibitors have been synthesised, for example those which are believed to contain potential angiotensin converting enzyme (ACE) inhibitors. In any case, libraries of potential compounds can be made using combinatorial chemistry techniques. It will be appreciated that the library of compounds may be a library of peptides, some of which bind to any suitable component such as the GFBP or the GFBP protease. The screening method or assay of the invention may be carried out using purified or semi-purified (ie cell-free) components, or it may be carried out using whole cells. Purified or semi-purified components may be obtained using biochemical methods, or it may be more convenient to make use of recombinantly-produced components. For example, as noted above, cDNAs are available which encode the GFBPs, the GFBP proteases and other useful components such as serine protease inliibitors (serpins) and growth factors. Genetic engineering techniques which are well known in the art may be used to express the polypeptide components. The GFBP may be in the extracellular matrix or it may be cell free. In a preferred embodiment, the method comprises the steps of providing a growth factor binding protein and a protease which is capable of proteolytically-degrading the binding protein, and determining whether the compound modulates the proteolysis of the binding protein by the protease. Conveniently, the assay comprises (1) a means for detecting the proteolysis of the growth factor binding protein (for example, by monitoring its cleavage), and (2) recombinant or purified binding protein protease, and the result (ie proteolysis of the binding protein) is measured in the presence or absence of the test compound. In this assay, compounds which increase the activity of the protease may be selected by their ability to lead to greater proteolysis of the GFBP in their presence compared to their absence. Compounds which decrease the activity of the protease may be selected by their ability to lead to less proteolysis of the GFBP in their presence compared to their absence. Proteolysis of the binding protein may be measured using any convenient method. For example, antibody-based methods such as ELISA may be used where the antibody can distinguish proteolysed and non-proteolysed GFBP; alternatively, radioligand blotting may be used, for example using radiolabelled growth factor, to determine when the GFBP has been proteolysed so as to no longer bind the growth factor, or to have reduced ability to bind the growth factor. High-throughput drug screening has traditionally involved assays based on in vitro cytotoxicity or cytostasis (Weinstein et al (1997) Science 275, 343-349), but this approach has been heavily criticised in terms of its efficacy (Cohen & Diamond (1986) Nature 323, 488-489; Editorial (1987) Nature 326, 729; Touchette (1991) J NIH Res 3, 41-44). Since in vivo processes such as bone density maintenance, hair growth, tissue fibrosis, blood vessel formation and tumour cell proliferation are likely to involve molecular interactions which are not purely mitogenic between different cell types, we describe here a panel of related methods for rational drug discovery based on drug effects involving an intermediary group of extracellular molecules which regulate growth factor action (GFBPs). By identifying and developing drugs which act selectively within the signalling pathway upstream of cell metabolism and growth 'decision- points' , it is submitted that relatively non-toxic and disease-specific ('tamoxifen-like') reagents with a better therapeutic index than many existing drugs will be systematically obtainable. It is particularly preferred if the growth factor binding protein present in the assay is any one of a binding protein which binds to a growth factor selected from any of polypeptide growth factors which are capable of binding transmembrane kinases; cytokines which are capable of binding hetero-oligomeric non-catalytic cytokine receptors; peptide hormones which are capable of binding seven-transmembrane-domain G-protein coupled receptors; and neurotransmitters which are capable of binding ligand-gated ion channels. It is particularly preferred if the growth factor binding protein present in the assay is any one of a binding protein which binds epidermal growth factor, transforming growth factor beta, erythropoietin, growth hormone, an interleukin, an interferon, platelet-derived growth factor BB, basic fibroblast growth factor, and insulin-like growth factors I and II. The following Table describes growth factors, GFBPs and BPPs, involved in various disease states, and it also describes protease inhibitors. Table Potential applications of the invention Some of the diseases in which the invention could potentially be used to identify candidate drugs include the following: GF GFBP BPP Prot. In. Disease Rxi Rxt IGFs IGFBP3 PSA uCT prostate Ca PSA (uPA) " X acromegaly X IGFBP3/4 X/Y osteoporosis X/Y IGFBP3 X cachexia X " X old age X " X liver met's X X TRP APP Alzheimer ' s APP HB-EGF HBSG TRP αlAT emphysema TRP bFGF peπecan X diabetic X retinopathy, Crohn's/UC, Kaposi's Sa TGF-β LTBP uPA, PAI-1 cancers, uPA PAI- 1 biglycan trans- fibroses, syndecan glutaminase transplant et al rejection EGFBP* wound EGF healing BP " endoghn X HHT X " decoπn X colon Ca X PDGF collagen MMP2 TIMP- 1 scleroderma TIMP # IV MMP9 atheroma MMP 2/9 EGF pro-EGF EGFBP acne, EGF scarring BP male pattern EGF baldness BP MSP pro-MSP EGFBP wound EGF healing BP HGF pro-HGF uPA HAI Gl Ca, etc uPA q In this context EGFBP acts as an activator, rather than an inliibitor, of BPP function. Abbreviations: GF growth factor GFBP GF binding protein BPP GF binding protein protease Prot. In. GFBP protease inhibitor Rxi drug target to be inhibited RxT drug target to be potentiated uPA uroplasmmogen activator (urokinase) cxCT alpha-chymotrypsinogen Ca cancer X as-yet-unidentified molecule TRP trypsin APP β-amyloid precursor protein HB-EGF heparin-binding epidermal growth factor HBSG heparin-binding sulphated glycoprotein(s) αlAT alpha- 1-antitrypsin bFGF basic fibroblast growth factor (FGF-2) UC ulcerative colitis Sa sarcoma TGF-β transforming growth factor-beta LTBP latent TGF-β-binding protein (complex) PAI-1 plasminogen activator inhibitor- 1 EGFBP EGF binding protein HHT hereditary haemorrhagic telangiectasia (type I) PDGF platelet-derived growth factor MMP matrix metalloprotease TIMP tissue inliibitor of metalloprotease MSP macrophage stimulating protein HGF hepatocyte growth factor HAI HGF activator inhibitor Gl gastrointestinal (eg stomach, pancreatic) The method or assay of the invention may be a cell based assay. The test compound may be contacted with a whole cell which expresses a GFBP and it is determined by a suitable method (such as antibody binding or growth factor binding) whether the GFBP has been modulated by the presence of the test compound, for example , whether its amount has increased, or its function altered, or its strucmral integrity altered. As noted, the first aspect of the invention provides a method of identifying or developing a compound which is useful in treating a growth factor- dependent disease, the method comprising the step of determining whether the compound modulates binding of the growth factor to the growth factor binding protein. Typically, the growth factor and the GFBP are contacted in the presence or absence of the test compound, and the effect of the test compound on the binding of the growth factor and the binding protein is determined. The test compound may enhance the binding of the growth factor and the binding protein; or the test compound may inhibit or prevent the binding of the growth factor and the binding protein. The binding of growth factor and binding protein may be determined using any suitable method. Suitable methods, such as radioligand binding, are well known in the art. Compounds which enhance the binding of growth factor and GFBP will have the effect of reducing the free growth factor; whereas compounds which inhibit the binding of growth factor and GFBP will have the effect of increasing the free growth factor. As discussed above, one growth factor-dependent disease is prostate cancer, and the method of the invention has proved particularly useful in identifying compounds which may be useful in treating, ameliorating or preventing prostate cancer. In particular, compounds identified using the methods of this aspect of the invention may be particularly useful in treating, ameliorating or preventing prostate cancer which has metastasised to the bone. Prostate specific antigen (PSA) is a protease which proteolytically- degrades insulin-like growth factor binding protein-3 (IGFBP-3). Thus, in a preferred embodiment in order to identify compounds potentially useful in treating, ameliorating or preventing prostate cancer, the assay includes IGFBP-3 as the growth factor binding protein, and, optionally, prostate specific antigen as the protease. In this screening method or assay, compounds which modulate IGFBP-3 amount, function or strucmral integrity, for example by decreasing the net effect of the IGFBP-3 protease on the GFBP are selected (including, but not restricted to, protease inliibitors). Compounds which have been identified by this method as potentially useful in the treatment of prostate cancer include the angiotensin converting enzyme (ACE) inhibitors lisinopril, captopril and enalapril. In contrast, as described above, the screening methods of the invention may be used to identify compounds which enhance or increase the activity of the GFBP protease. Such compounds are useful in the treatment of growth factor-dependent diseases in which it is desirable to release the growth factor from the GFBP (or prevent the growth factor from becoming sequestered). In relation to IGFBP-3, compounds which enhance or increase the activity of an IGFBP-3 protease may be useful in treating osteopaenic bone disease such as osteoporosis. It can be seen that the foregoing observations provide a novel and unobvious approach to the identification and/or development of new therapeutic agents, particularly those for the treatment or prevention of cancer or osteoporosis. However, it is proposed that any growth factor- dependent disease process, especially those involving known or suspected protease dysregulation, in particular growth factor binding protein protease dysregulation whether by up- or down-regulation of protein expression, ectopic expression, functional over- or underactivity, or abnormal changes in substrate abundance or form, is a candidate for this drug development/ screening strategy. In general terms, and making use of the foregoing methods of the invention as appropriate, the following steps may be employed: 1. Identify disease-linked protease (P; eg PSA) and/or protease substrate (S; eg IGFBP-3 or other GFBP) of interest within appropriate test system (eg animal model, LNCaP cells) or as appropriate to the disease; 2. Identify definitive assay (A) for commercially viable drug; eg inhibition of cancer cell growth (for cancer drug), bone densitometry (for osteoporosis drug), or as appropriate to the growth factor-dependent disease; 3. Treat cells/animal with candidate drug(s), and assay effects on P function using measurement of S; 4. Take 'positive' drugs from this assay, and re-test in ' A; 5. Take 'positive' , drugs from A, thereby creating lead compounds for further modification and repeated rounds of testing, or else early-phase drugs for clinical evaluation. (Note that 'functional' , inhibition or potentiation in this context does not necessarily imply a direct interaction between drug and protease, nor does it necessarily imply an effect on the catalytic activity of the protease; the mechanism of net effect is immaterial.) Hence, the identification or development of higher-specificity inhibitors or agonists of PSA and/or other GFBP proteases, for example, can be achieved without undue ingenuity by one of ordinary skill in the art using the foregoing steps. Notwithstanding the above, it may be preferred if the GFBP protease is not PSA or that the GFBP is not an IGFBP in the assays and methods of the invention. The data presented in the Examples provide evidence that the erstwhile 'tumor marker' PSA is in fact a valid therapeutic drug target. Hence, protease expression by tumors may indicate complex in vivo growth loops which are difficult to test or confirm using conventional in vitro single-cell test systems. Systematic screening of established or experimental drugs for functional interaction with putative disease-associated proteases (eg by measuring in vitro changes in substrate composition or abundance) should thus provide a way of 'shortlisting' compounds for assessment in definitive assays, eg cell growth. Named drugs were screened for their ability to modulate in vitro expression and/or proteolysis of the target protease substrate, IGFBP-3. Using a standard radioligand blotting assay to detect IGFBP-3 in conditioned media of the PSA-expressing androgen-sensitive LNCaP prostate cancer cell line, two drugs were identified which clearly increased detectable levels of intact IGFBP-3 within the medium. These drugs, having been functionally characterized as IGFBP-3 protease inhibitors in this way, were then tested for their ability to affect androgen-stimulated LNCaP cell growth. This led to the finding, unexpected from the prior art, that both of the IGFBP-3 protease inhibitors identified in this manner selectively inhibited androgen-dependent LNCaP cell growth. The extent of this inhibition was quantitatively similar to that induced by addition to the medium of recombinant IGFBP-3. This result implies that pharmacologic inhibitors of PSA. for example, may be effective and highly specific cytotoxic agents in the management of prostate cancer, perhaps including hormone- resistant cancers which are currently refractory to most drugs. There are various uses for drugs interacting with PSA or other IGFBP- proteases in an inhibitory or agonistic (activity-enhancing) fashion, for example inhibitors of PSA, or other GFBP proteases may be used in palliation of metastatic bone disease in prostate cancer; treatment of noi - bony metastatic disease in prostate cancer; treatment of local or regional prostate cancer; neoadjuvant treatment of prostate cancer prior to local therapy; adjuvant prostate cancer treatment following local therapy; prevention of prostate cancer development or progression; prevention or treatment of other cancers in which IGFBP-3 or other GFBP functions as a tumor-inliibitory molecule. Agonists of PSA, or other GFBP proteases, may be used for prevention or treatment of cancers in which IGFBP-3 or other GFBP enhances tumor growth or progression; primary prevention of osteoporosis in susceptible individuals; secondary treatment of bone loss in affected individuals. Thus, in various aspects, the present invention provides the development or identification of: (a) drugs inhibiting the effects of PSA or other GFBP proteases: for therapeutic use in prostate cancer patients with metastatic bone disease; for therapeutic use in prostate cancer patients with metastases affecting sites other than bone; for therapeutic use in patients with local or regional prostate cancer; for adjuvant therapeutic use in prostate cancer patients prior to the development of clinically evident metastases; for neoadjuvant therapeutic use in prostate cancer patients prior to local therapies for prostate cancer; for prevention of prostate cancer: and for prevention or treatment of non-prostatic cancers in which the target GFBP (protease substrate) acts as a tumor-inhibitory molecule; (b) drugs potentiating the effects of PSA or other IGFBP-3 proteases: for prevention or treatment of cancers in which the target GFBP (protease substrate) enhances tumor growth or progression; for therapeutic use in the prevention of osteoporosis; and for therapeutic use in established or incipient osteoporosis; (c) drugs inhibiting or potentiating the effects of PSA or other GFBP proteases: known or suspected to affect the symptoms, clinical manifestations or natural history of diseases other than cancer or osteoporosis; known or suspected to affect the symptoms, clinical manifestations or natural history of diseases including cancer and osteoporosis; known or suspected to affect the symptoms, clinical manifestations or natural history of diseases including cancer and osteoporosis; and for sale to the research or diagnostics markets, whether in kit form or singly, for commercial or academic use, for in vitro or in vivo use. A second aspect of the invention provides a method of treating a growth factor-dependent disease, the method comprising the steps of administering to the patient an effective amount of a compound which modulates the amount, function or strucmral integrity of a growth factor binding protein (for example, though not necessarily, by altering the net effect of a protease upon such a growth factor binding protein). Preferably, the disease is one involving known or suspected protease dvsregulation, or one in which it is desirable to modulate the activity of a GFBP protease. Preferably, the compound is selective for its target. For example, it is preferred that if the compound is a protease inhibitor it selectively inhibits the protease which is capable of inactivating GFBP associated with the disease in preference to other proteases. However, as is clear from the observations below in relation to angiotensin converting enzyme (ACE) inliibitors, useful protease inhibitors may inhibit not only the GFBP protease but other proteases. In a preferred embodiment of the invention the compound increases the activity of the protease. This is useful in diseases in which it is desirable to increase the proteolysis of a GFBP, increased proteolysis of which may lead to release of the growth factor bound by the GFBP or it may prevent sequestration of the growth factor by the GFBP. In relation to IGFBP-3, which binds insulin-like growth factor, it is desirable to activate the IGFBP-3 protease in the treatment of osteopaenic bone disease, such as osteoporosis. In a further preferred embodiment of the invention, the compound decreases the activity of the protease. This is useful in diseases in which it is desirable to decrease the proteolysis of a GFBP, decreased proteolysis of which may lead to retention of the growth factor by the GFBP, or it may aid its sequestration. In relation to IGFBP-3, which binds insulin-like growth factor, it is desirable to inhibit the IGFBP-3 protease in the treatment of prostate cancer, particularly prostate cancer which has metastasised to the bone. PSA is a protease which acts on IGFBP-3; thus, it is desirable if the compound is one which inhibits PSA. The compounds which are useful in this aspect of the invention may directly or indirectly modulate the amount, function or strucmral integrity of the GFBP as is described above in relation to the first aspect of the invention. It is preferred that the compound directly modulates the GFBP protease and, in relation to the decrease of protease activity, it is preferred if the compound directly inhibits the protease. As is described in the Examples, we have identified a number of inhibitors which inhibit GFBP protease activity. These inhibitors include, but are not limited to, angiotensin converting enzyme (ACE) inhibitors (including lisinopril, captopril, and enalapril), and other protease inhibitors such as dichloroisocoumarin (DCI). Other available ACE inhibitors include alacepril; benazepril; ceronapril; cilazapril; delapril; enalaprilat; fosinopril; imidapril; libenzapril; moexipril; moveltipril; pentopril; perindopril; pivopril; quinapril; ramipril; spirapril; temocapril; teprotide; trandolapril; zofenopril. Metalloproteinase inhibitors such as marimostat and batimastat may be useful. It will be appreciated that an effective amount of the compound is an amount effective to prevent, reduce, ameliorate or palliate the disease to a useful extent. Preferably, the amount of the compound administered does not produce undesirable side effects. Notwithstanding the above, it may be preferred if, when the disease is prostate cancer, the compound is not defined generally as a protease inhibitor. This, however; is without prejudice to the use of specific protease inhibitors being part of the invention, for example angiotensin converting enzyme (ACE) inhibitors. A third aspect of the invention provides a method of treating prostate cancer, the method comprising administering to the patient an effective amount of a compound that modulates the amount, function or strucmral integrity of insulin-like growth factor binding protein-3. Preferably, the compound acts on the GFBP protease (most preferably directly) and, most preferably, the compound inhibits the enzyme activity of the protease. In a particularly preferred embodiment the compound is a protease inhibitor including any suitable ACE inhibitor. It is particular to use this aspect of the invention to treat prostate cancer which has metastasised to the bone. A fourth aspect of the invention provides a method of treating osteopaenic bone disease such as osteoporosis, the method comprising administering to the patient an effective amount of a compound that increases the activity of a protease which protease is capable of inactivating a insulin-like growth factor binding protein-3. Activation of this protease may deplete the bone of IGFBP-3 which may, in mrn, lead to strengthening of the bone. 2S The invention also includes a method of treating osteopaenic bone disease such as osteoporosis, the method comprising administering to the patient an effective amount of a compound that increases the activity of a protease which protease is capable of inactivating a insulin-like growth factor binding protein-3. Without prejudice to the mode of action of an ACE inliibitor, a further aspect of the invention provides a method of treating prostate cancer, especially prostate cancer metastasised to the bone, with an ACE inliibitor. Thus, a fifth aspect of the invention provides a method of treating prostate cancer, the method comprising administering to the patient an effective amount of an angiotensin-converting enzyme inliibitor. A sixth aspect of the invention provides the use of a compound which modulates the amount, function or structural integrity of a growth factor binding protein in the manufacture of a medicament for treating a growth factor-dependent disease. A seventh aspect of the invention provides the use of a compound that modulates the amount, function or structural integrity of a growth factor binding protein in the manufacture of a medicament for treating prostate cancer. An eighth aspect of the invention provides the use of an angiotensin- converting enzyme inliibitor in the manufacture of a medicament for treating prostate cancer. The ACE inhibitor, such as lisinopril, may be administered with a daily dose of 10 mg. Captopril may be used at 25 mg twice-daily, provided that hypotension does not occur. The ACE inhibitor may be administered orally. It is particularly useful to administer the ACE inhibitor when bone metastases are present, or in adjuvant therapy of prostate cancer. A ninth aspect of the invention provides the use of a compound that modulates the amount, function or strucmral integrity of a growth factor binding protein in the manufacture of a medicament for treating osteopaenic bone disease, such as osteoporosis. From the foregoing, and from the following Examples, it will be seen that my data indicate an autocrine effect of the PSA/IGFBP-3 system on androgen stimulated LNCaP cells, ie in addition to the in vivo paracrine effects inferred from the bone densitometric data, and on my data indicating that peripheral (normal) bone density is linked to high circulating levels of the IGFBP-3 protease PSA, and that drugs affecting the function of such proteases can be readily identified using the assay approach described. Relevant to this method of identification, I note the study of Valentinis et al (1995) Molecular Endocrinology 9, 361-367 in which the growth- inliibitory properties of IGFBP-3 are shown to be independent of its ability to modulate signalling via the IGF receptor system. Consistent with this, we have confirmed that, notwithstanding the demonstrated growth- inliibitory effects of IGFBP-3 on LNCaP cells (Figures 7 and 10), these cells are not growth-stimulated by IGFs (Figure 15) whereas in contrast, androgen-independent PC-3 cells appear highly IGF-sensitive yet resistant to PSA (Figure 16) . These findings emphasize that the invention is wholly endpoint-based rather than mechanism-based, with the sole criterion for drug delivery being a drug-inducible alteration in the amount, strucmral integrity, or function (eg growth factor binding) of the GFBP being assayed. The prior art does not replicate or anticipate the new and unexpected results listed herein. No publication of which I am aware has produced in vivo evidence confirming a role for PSA or other IGFBP proteases as drug targets in either prostate cancer or osteoporosis. Indeed, any mention of this notion is conspicuously absent from recent editorials and comprehensive reviews relating to PSA 2 ' 38 ' 40 , insulin-like growth factor 21 or the treatment of prostate cancer ' . Numerous signalling molecules distinct from the IGFs have been supported in the literature as proximate activators of osteoblasts within sclerotic prostate cancer metastases. These include endothelin-1 , parathyroid hormone-related protein 4" , transforming growth factor-β 16 , basic fibroblast growth factor 48 , and prostatic acid phosphatase 49. IGFBP-3 is only one of numerous in vitro and in vivo PSA substrates identified and studied. The main substrate of PSA is acknowledged to be seminogelin, fibrous protein within semen, cleavage of which is required for normal liquefaction of seminal vesicle secretions. In vitro PSA substrates include insulin, interleukin-2, fibrinogen, ovalbumin , parathyroid hormone-related protein 15 , transforming growth factor-β 16 , none of which yield cleavage products known to be mitogenic for prostate cancer cells (TGF-β is inhibitory). Hence, the finding that IGFBP-3 is capable of being cleaved in vitro by PSA does not establish the in vivo significance of this phenomenon. Proteases other than PSA have been implicated in the osteoblastic phenotype. These include urokinase 10~12 and cathepsin D, the latter of which has been shown to cleave IGFBP-3 13 . Still other endogenous proteases, such as matrix metalloproteases I and III. can degrade IGFBP- 3 . Moreover, catabolism of IGFBP-3 appears inducible in numerous other scenarios including diabetes mellitus 2 and physical distress 28 . Hence, the possibility that IGFBP-3 levels may be low in some diseased prostate " , or in the sera of patients with metastatic disease " , does not imply an obvious causative link between PSA expression and the progression of prostate cancer. Many references in the literature suggest conclusions which at first glance appear to contradict the present invention. For example, inhibition of PSA expression by the growth factor-interactive drug suramin may not be associated with prostate cancer cell growth inhibition , again suggesting lack of a cause-and-effect relationship between PSA and growth. Similarly, the concentration-dependence of androgen-stimulated LNCaP cell growth differs markedly from that of PSA induction 23 . With respect to the significance of IGFBP-3, the extent of IGFBP-3 proteolysis within normal and neoplastic human tissues does not correlate quantitatively with on PSA expression levels ; moreover, the finding of reduced intraprostatic IGFBP-3 in this study appears incompatible with prior art indicating full 17 inhibition of catalytic activity within this organ . Finally, a substantial body of literature indicates that IGFBP-3 potentiates rather than inhibits IGF-1 activity 19 ' 21 . Selective reference to the literature might suggest that PSA is capable of acting as a persistent 'IGF amplifier' , ie an oncogene. In fact, there is no ^2 published data confirming PSA to be an oncogene. On the contrary, studies from the breast cancer literamre implicate PSA as a mor suppressor , the expression of which is associated with good prognosis 6 . Consistent with this possibility, tumor- suppressive activity has been reported for many other proteases 51 ' 52 , thus indicating that protease activity alone cannot be construed as oncogenic, irrespective of short-term in vitro effects on growth factor action or availability. PSA-like molecules are not linked to the growth of prostate cancer in rodents . This finding indicates, for example, that 'knockout' of PSA- like rodent molecules in transgenic mice would have no effect on in vivo prostate cancer growth. Mice bear a strong phylogenetic and physiologic resemblance to humans, making this mammalian system the main model for human diseases. Hence, this negative finding casts substantial doubt on the centrality of PSA as a pathogenetic mediator of prostate cancer, tending rather to support the traditional view of PSA as a purely adjunctive molecule which happens to act as a mmor marker. The invention will now be described in more detail with reference to the following Examples and Figures wherein: Figure 1. Comparison of metastatic bone disease in two patients with prostate cancer differing in primary tumor PSA expression phenotype. Compared with the classical osteosclerotic changes of metastatic PSA- positive prostate cancer (case #1 , left), the radiograph on the right (case #2) shows virtually complete osteolysis of the pelvic ischium and pubis. Immunohistochemistry of the associated primary prostate cancers confirmed abundant PSA in case 1 , but undetectable PSA in lane 2. Figure 2. Comparison of PSA and serum alkaline phosphatase (SAP; a marker of osteoblastic activity) in eight bone metastases from prostate cancer. Figure 3. Relationships between PSA, osteoblastic reaction, and peripheral bone density in a consecutive cohort of patients with metastatic prostate cancer. A, Serum alkaline phosphatase (SAP, on the ordinate) as a function of plasma PSA (on the abscissa). B, SAP as a function of tibial bone density (TBD, on the abscissa). TBD was determined by dual X-ray absorption (DXA) bone densitometry; isotope bone scanning confirmed lack of disease involvement in this site in each case. C, Plasma PSA (represented as a logarithmic function) related to lesional blastic index (BI, on the abscissa) . Prostate cancer patients presenting for the first time with symptomatic bone metastases were staged using serum PSA and bone scan. Scintigraphically detected lesions were evaluated by plain X-ray, and radiographic lesions were further characterized using a Victoreen model 07-444 densitometer. For each assessable metastatic lesion, X-ray densitometry was undertaken of adjacent normal bone as determined by lack of involvement on bone scan. The BI for a given patient was then defined as the mean density of lesional bone expressed as a proportion of adjacent normal bone density; hence, lytic lesions were characterized by BI < 1 ; sclerotic lesions, BI > 1 , and radiographically occult (but bone scan-positive) lesions, BI= 1. Wherever possible, multiple lesions were measured to yield an average patient BI. This approach was validated by DXA bone densitometry using a Lunar DPX densitometer in a prospective series of patients, which yielded lesional density enhancements quantitatively similar to the BI. Figure 4. Radioligand blotting of plasma IGFBP-3 in the presence and absence of PSA. A, Dilution series of seminal plasma (SP) in normal serum from a volunteer. Control serum, lane 1 ; doubling increments in SP concentration, lanes 2-5. B, Serum from prostate cancer patients without plasma PSA elevation (lane 1) or with plasma PSA elevation (lane 2). Figure 5. IGFBP-3 content of bone metastases from patients with breast cancer (BC) or prostate cancer (PC). A, Radioligand blot of IGFBP-3 content in breast cancer metastases (lanes 2, 3) and prostate cancer metastases (lane 4, 5) and normal bone marrow (BM, lane 6); recombinant (control, C) IGFBP-3 is shown in lane 1. B, Total protein content of the tumour samples as determined by Coomassie blue gel staining of duplicate ly sates; the position of albumin is indicated. C, PSA immunohistochemical staining of one each of the breast (BC) and prostate (PC) metastases. Figure 6. Growth factor content of metastatic bone samples from five prostate cancer (PC) and eight breast cancer (BC) patients as determined by ELISA. A, Tumour content of the growth factors FGF-2, TGF-βl , IGF-1 , TGF-α (transforming growth factor-alpha), and EGF. B, Validation of the tumour content using either PSA (left) or anti-cytokeratin (CK; right). Figure 7. Relationship between PSA, IGFBP-3 levels, and androgen- dependent growth of LNCaP human prostate cancer cells. A, Induction of cellular PSA content in response to synthetic androgen (R1881) as determined by Western blotting. Lane 1 , control cells; lane 2, treated for 24h with R1881 ; lane 3, treated for 2 days; lane 4, treated for 3 days. B. Growth curve of control and androgen-stimulated cells. C, Cell growth after three days as determined by colorimetric (sulphorhodamine) assay in control cells (C), IGFBP-3-treated cells (BP3), androgen-treated (R), and androgen-plus IGFBP-3-treated cells (R + BP3). D, Effect of the PSA inhibitor 3,4-dichloroisocoumarin (DCI) on IGFBP-3 levels in androgen- treated LNCaP cell supernatants as determined by radioligand blotting. Lane 1 , recombinant IGFBP-3 alone; lane 2, charcoal-stripped serum from androgen-stimulated cells; lane 3, effect of DCI treatment on a cell sample otherwise identical to lane 2. E, Effects of DCI on androgen-dependent LNCaP cell growth. Samples were grown for three days, then counted. F, Radioligand blot of IGFBP-3 concentration in LNCaP cell supernatants. Lane 1 , untreated cell medium at time t = 0; lanes 2 and 3, untreated cell medium at t = 24h; lane 4, cells treated for 24h with androgen; lane 5, cells treated for 24h with the angiotensin-converting enzyme (ACE) inhibitor lisinopril; lane 6, cells co-treated with androgen and lisinopril for 24h. G, Growth of LNCaP cells in response to androgen and/or lisinopril (LIS). Figure 8. Radiographic and immunohistochemical comparison of metastatic bone disease in two patients with prostate cancer. Compared with the classical osteosclerotic changes of metastatic PSA-positive prostate cancer (case #1, left), the radiograph on the right (case #2) shows virtually complete osteolysis of the pelvic ischium and pubis. Immunohistochemistry of the associated primary prostate cancers confirms abundant PSA in case 1 , but undetectable in case 2. Table 1. Non-parametric analysis of prostate cancer patient subgroups defined by (i) the serum PSA level at the time of first metastatic presentation, and (ii) the osteosclerotic index (OSI). Prostate cancer patients presenting for the first time with symptomatic bone metastases were staged using serum PSA and bone scan. Scintigraphically detected lesions were evaluated by " plain X-ray, and radiographic lesions were further characterized using a Victoreen model 07-444 densitometer. For each assessable metastatic lesion, X-ray densitometry was undertaken of adjacent normal bone as determined by lack of involvement on bone scan. The OSI for a given patient was then defined as the mean density of lesional bone expressed as a proportion of adjacent normal bone density; hence, lytic lesions were characterized by OSK 1 ; sclerotic lesions, 0SI > 1 , and radiographically occult (but bone scan-positive) lesions, OSI = l . Wherever possible, multiple lesions were measured to yield an average patient OSI. This approach was validated by dual X-ray absorption (DXA) bone densitometry using a Lunar DPX densitometer in a prospective series of patients, which yielded lesional density enhancements quantitatively similar to the OSI. Statistical calculations based on two PSA cut-offs (10 or 20 ng/ml) are shown. Figure 9. Interrelationship between IGFBP3 (BP3) and PSA in vitro and in vivo. A, PSA cleavage of IGFBP3 in vitro and in vivo. Effect of doubling concentrations (lanes 2, 62.5 ng/ml; lane 3, 125 ng/ml) of purified PSA on the recombinant ~ 40-kDa IGFBP3 protein as detected by 125 I-IGF2-ligand blotting. B, Radioligand blot demonstrating IGFBP3 levels in patients with primary (lane P) and metastatic (lane M) prostate cancer, showing reduction of the 40-kDa band and an increased intensity of a ~ 33-kDa band which corresponds to the size of IGFBP2. C, Relationship between PSA and IGFBP3 expression levels in breast and prostate cancer bone metastases. Radioligand blot (top panel) of metastatic bone tissue from breast cancer (BC; lanes 2,3) and prostate cancer (PC; lanes 4,5) patients compared with control (C; lane 1 , recombinant IGFBP3) and normal bone marrow (BM; lane 6). Coomassie-stained gels are shown below, confirming similar amounts of total protein loaded, while anti-cytokeratin (CK) immunoblots and PSA ELISAs confirm the neoplastic nature of the samples. Figure 10. Functional relationship between PSA, IGFBP3, inhibitors of PSA-dependent proteolysis, and androgen-dependent LNCaP cell growth. R1881, a synthetic androgen, was used to simulate cells. A, Androgen- dependent cell PSA expression (upper panel), and supernatant IGFBP3 concentrations (BP3; lower panel). Samples were stimulated for 0 (lane 1), 1 (lane 2), 2 (lane 3) or 3 (lane 4) days prior to lysis and PSA immunoblotting or 125 I-IGF ligand blotting; data points for the latter were calculated as the percentage of androgen-stimulated to control IGFBP3 levels as quantified by X-ray densitometry of radioligand blot 40 kDa bands. B, Inhibition of androgen-dependent LNCaP cell growth by IGFBP3 (upper panel) and the PSA inhibitor DCI (lower panel). Cells were grown for 3 days with or without addition to the medium of R1881 (R; 3 x 10 "10 M; lanes 3, 4) plus or minus 50 nm IGFBP3 (BP3; lanes 2, 4) for the same period (top); cells were grown for 3 days with or without addition to the medium of R1881 (R; 3 x 10 " '°M; lanes 3, 4) plus or minus the PSA antagonist dichloroisocoumarin (DCI, 10 "6 M; lower panel; lanes 2, 4) for the same period. Figure 11. Mitogenic effects of adding recombinant PSA or androgen (R1881) to the supernatant of LNCaP cells. PSA was added where shown at an initial concentration of 100 ng/ml, and cells were allowed to grow for 72 h. 3S Figure 12. Effect of the ACE inhibitor lisinopril on IGFBP-3 levels in control and androgen-treated LNCaP prostate cancer cells. A, In vivo treatment: radioligand blot of IGFBP-3 concentration in LNCaP cell supernatants. Lane 1 , untreated cell medium at time t = 0; lanes 2 and 3, untreated cell medium at t = 24h; lane 4, cells treated for 24h with androgen; lane 5, cells treated for 24h with the angiotensin-converting enzyme (ACE) inhibitor lisinopril; lane 6, cells co-treated with androgen and lisinopril for 24h. B, In vitro treatment. Control buffer containing IGFBP-3 co-incubated with purified PSA (lane 1) was combined with either the PSA inhibitor DCI (10 "6 M, lane 2; 10 "8 M lane 3) or lisinopril (10 " M, lane 4; 10°M, lane 5) prior to termination of the reaction after 60 mins, electrophoresis, and 3 I-IGF radioligand blotting. Figure 13. Effects of lisinopril and DCI on androgen-dependent LNCaP cell growth. Cells were treated with either DCI (10 " M) or lisinopril (10 "6 M) as shown for 3 days. Figure 14. Radioligand ( ^I-EGF) ligand blotting to detect EGF-binding proteins. Cells as designated were lysed, electrophoresed, and transferred to membranes. The membrane-bound protein lysates were then probed using radioiodinated EGF (Amersham), yielding a single hot spot in lysates from EGFR-overexpressing A431 human squamous cancer cells. Figure 15. Effects of IGF-1 and PSA on growth of androgen-dependent LNCaP human prostate cancer cells. Effects of IGF-1 treatment on growth of androgen-treated and -untreated LNCaP cells are shown in the upper panel, and effects of PSA (100 ng/ml) treatment on growth of androgen-untreated LNCaP cells in the lower panel. Figure 16. Effects of IGF-1 and PSA on growth of androgen-independent PC-3 human prostate cancer cells. Effects of IGF-1 on PC-3 cell growth over 3 days is shown in the upper panel, and effects of PSA (200 ng/ml, 3 days) on PC-3 cell growth in the lower panel. Example 1: Selective loss of IGFBP-3 in osteoblastic bone metastases from prostate cancer implicates a protease-dependent mechanism of paracrine tumor growth The unusual sclerotic appearance of bone metastases from prostate cancer is unexplained. One possibility is that osteosclerosis occurs due to excess peritumoral activity of osteoblast mitogens, of which the most abundant are the insulin-like growth factors (IGFs). Since prostatic metastases are also distinguished by secretion of prostate-specific antigen (PSA) - a serine protease which cleaves the major insulin-like growth factor binding protein IGFBP3 - I sought to clarify the relationship between PSA function, IGFBP3 availability, and prostate cancer behavior in vivo and in vitro. A retrospective clinical analysis revealed an association between 'low-PSA' ( < 20 ng/ml) metastatic prostate cancer and osteolytic bone lesions (p < 0.0001). Direct examination of bony lesions revealed that IGFBP3 levels in breast cancer metastases were similar to those from benign marrow reamings, whereas PSA-positive prostate cancer metastases were IGFBP3 deficient. Consistent with a mitogenic role for IGFBP3 proteolysis, androgen-stimulated growth of PSA-expressing LNCaP cells was antagonized by treatment with recombinant IGFBP3 or a pharmacologic PSA inhibitor. Although indirect, these findings suggest that the characteristic osteoblastic phenotype of prostatic metastases may reflect enhanced IGF bioavailability due to the action of IGFBP3 proteases including (though not necessarily restricted to) PSA. Importantly, such proteases may represent novel therapeutic targets for the palliation of hormone-refractory prostate cancer. Materials and Methods Clinical studies Prostate cancer patients presenting for the first time with symptomatic bone metastases were staged using serum PSA and bone scan. Scintigraphically detected lesions were evaluated by plain X-ray, and radiographic lesions were further characterized using a Victoreen model 07-444 densitometer. For each assessable metastatic lesion, X-ray densitometry was undertaken of adjacent normal bone as determined by lack of involvement on bone scan. The osteosclerotic index (OSI) for a given patient was then defined as the mean density of lesional bone expressed as a proportion of adjacent normal bone density; hence, lytic lesions were characterized by OSI < 1; sclerotic lesions, OSI > 1 , and radiographically occult (but bone scan-positive) lesions, OSI = 1. Wherever possible, multiple lesions were measured to yield an average patient OSI. This approach was validated by dual X-ray absorption (DXA) bone densitometry using a Lunar DPX densitometer in a prospective series of patients, which yielded lesional density enhancements quantitatively similar to the OSI. Metastatic bone marrow deposits were snap-frozen from surgical specimens of patients with advanced breast or prostate cancer undergoing repair of pathological fractures. Tumor content was verified by immunohistochemical analysis. An aliquot of each mmor was disrupted under liquid nitrogen using pestle and mortar, resuspended in ice-cold lysis buffer (1 % NP-40, 10% glycerol, 50mM sodium fluoride, lOmM Na 2 HPO 4 .H 2 0, lOmM NaH 2 PO 4 .H 2 0, lOmM NaPPi, ImM sodium orthovanadate, and 1 μg/ml each of the protease inhibitors antipain, aprotinin and leupeptin), sonicated twice for 30 sees then centrifuged. Tumor content of each supernatant was verified using either immunoblotting for cytokeratins (breast cancer) or ELISA for PSA (prostate cancer) in conjunction with spectrophotometry and Coomassie staining for protein quantification. In vitro studies LNCaP cells were obtained from American Type Culture Collection (Rockville, MD), maintained as stocks in RPMI-1640 medium with 10% fetal calf serum, antibiotics and glutamine, and transferred to medium containing 5% dextrancoated-charcoal double-stripped serum 5 days prior to hormone stimulation experiments. The synthetic androgen methyltrienolone R1881 (metribolone; a gift of Roussel. France) was used at 3 x 10 "10 M for growth experiments. Cell proliferation was measured using a sulphorhodamine protein-binding assay (Skehan, P. , Storeng, R. , and Scudiero, D. , et al (1990) New calorimetric cytotoxicity assay for anticancer drug screening, J Natl Cancer Inst. 82, 1107-1112). Antibodies to PSA and cytokeratins were purchased from R&D Systems (Abingdon, UK). Recombinant IGFBP3 was purchased from Biogenesis (DSL, Poole UK), 125 I-IGF2 for radioligand blotting studies to detect IGFBP3 (Hossenlopp, P. , Seurin, D. , Segovia-Quinson, B. , Hardouin, S. , Binoux, M. (1986) Analysis of serum insulin-like growth factor binding proteins using western blotting: use of the method for titration of binding proteins and competitive binding studies, Anal Biochem. 154, 138-143) from Amersham (Bucks, UK), and the PSA inhibitor dichloroisocoumarin (DCI; ref. 24) from Sigma. Proteins were electrophoresed under denaturing non-reducing conditions, transferred to nitrocellulose, then subjected to immunoblotting or radioligand blotting using standard protocols. Results Relationship between serum PSA levels and radiographic osteosclerosis An X-ray of a prostate cancer patient who relapsed with widespread osteolytic lesions is shown in Figure 8 (case #2; top right), revealing striking dissolution of the pubic and ischial rami in the pelvis; this appearance contrasts with that of a typical prostate cancer patient with metastatic disease associated with radiographic sclerosis of the entire pelvic girdle (case #1 , top left). The former case was associated with a 'low' PSA level ( < 10 ng/ml) relative to that expected for this extent of disease; immunopathologic review confirmed the unusual PSA-negative nature of the original prostate cancer (Fig. 8, bottom right), consistent with this primary origin. Since this observation raised the hypothesis of a causal link between PSA expression and osteoblastic reaction within metastatic lesions, a retrospective review was undertaken. A cohort of metastatic prostate cancer patients exhibiting a range of serum PSA levels at the time of first metastatic presentation were identified from the Charing Cross Tumor Marker Laboratory database; those with full staging documentation (PSA, X-rays, bone scan) at that time were characterized further. X-ray densitometry of lesional and adjacent normal bone, as defined by isotope scan correlation, was undertaken in this cohort (see Methods) and the resulting quantification used to generate a measure of lesional bone sclerosis ('osteosclerotic index' , OSI). Consistent with earlier studies (Soloway, M. S. , Hardeman, S. W. , Hickey, D. , Raymond, J. , Todd, B. , and Moinuddin, M. (1992) Stratification of patients with metastatic prostate cancer based on extent of disease on initial bone scan, Cancer 62, 195-202), this revealed no linear correlation between the height of the serum PSA and the OSI (r = 0.05; data not shown). These data were then reanalyzed using non-parametric comparison of subgroups defined by OSI ( > 1 or < 1) and PSA cutoffs of either 10 or 20 ng/ml - the latter being values popularly used to determine the need for bone scan in primary disease (Oommen, R. , Geethanjali, F. S. , and Gopalakrishnan, G. , et al (1994) Correlation of serum PSA levels and bone scintigraphy in carcinoma prostate, Br J Radiol. 67, 469-471 ; Morote, J. , Lorente, J. A. , and Encabo, G. (1996) Prostate carcinoma staging: clinical utility of bone alkaline phosphatase in addition to prostate specific antigen, Cancer 78, 2374-2378). This analysis (see Table) revealed highly significant clustering of osteolytic and nonsclerotic lesions in the 'low-PSA' patient cohorts (see Discussion). Table 1 (to accompany Fig 8) OSI≤ l OSI > l (n = 5) (n = 22) Serum PS A < 10 ng/ml 4 1 (« = 5) Serum PSA > 10 ng/ml 1 21 (n = 22) (X 2 = 15.4) ( < 0.0001) Serum PSA<20 ng/ml 5 1 (72 = 6) Serum PSA > 20 ng/ml 0 21 (n = 21) (X 2 = 21.5) (p < 0.0001) Table 1. Non-parametric analysis of prostate cancer patient subgroups defined by (i) the serum PSA level at the time of first metastatic presentation, and (ii) the osteosclerotic index (OSI; see Methods). Two PSA cut-offs (10 or 20 ng/ml) are used. Relative expression patterns of PSA and IGFBP3 in bone metastases Purified PSA efficiently cleaves the IGF binding protein IGFBP3 in vitro Fig. 9 A as reported elsewhere (Cohen, P. , Graves, H. , Peehl, D. M. , Kamarei, M. , Giudice, L. C , and Rosenfeld, R. G. (1992) Prostate- specific antigen (PSA) is an insulin-like growth factor binding protein-3 protease found in seminal plasma, J Clin Endoc Metab. 75, 1046-1053; Cohen, P. , Peehl, D. M. , Graves, H. , and Rosenfeld, R. G. (1994) Biological effects of PSA as an insulin-like growth factor binding protein- 3 protease, J Endocrinol. 142, 407-415); however, attempts using this approach to convert TGFβ to the active form in vitro as reported by others (Killian, C. S. , Corral, D. A. , Kawinski, E. , and Constantine, R. I. (1993) Mitogenic response of osteoblast cells to PSA suggests an activation of latent TGF-β and a proteolytic modulation of cell adhesion receptors, Biochem Biophys Res Comm. 192, 940-947) were negative (data not shown). Comparative 12D I-IGF2 ligand blotting of sera from a primary (P) prostate cancer patient and a patient with PSA-secreting metastatic (M) prostate cancer reveals reduced IGFBP3 and increased IGFBP2 Fig. 9B consistent with an earlier report (Kanety, H. , Madjar, Y. , and Dagan, Y. , et al (1993) Serum insulin-like growth factor-binding protein-2 (IGFBP-2) is increased and IGFBP-3 is decreased in patients with prostate cancer: correlation with serum prostate-specific antigen, J Clin Endoc Metab. 11 ' , 229-233). To determine the relative abundance of IGFBP3 in lytic and sclerotic bone metastases, I applied this radioligand binding assay to marrow reamings from pathologic fractures of patients with breast or PSA-positive prostate cancer; to minimize the risk of normal tissue admixture, only samples containing abundant cytokeratin or PSA respectively were analyzed. Four such metastatic specimens (two prostate, two breast) plus one aliquot of normal bone marrow were thus examined, revealing a striking relative loss of radioligand binding to IGFBP3 in the prostatic specimens (Fig. 9C); as expected, immunohistochemistry confirmed absent PSA expression in the breast metastases. In vitro relationship between androgen-inducible cell growth, PSA, and IGFBP3 LNCaP human prostate cancer cells exhibit increased growth in response to androgens (Lee, C , Sutkowski, D. M. , and Sensibar, J. A. , et al (1995) Regulation of proliferation and production of prostate-specific antigen in androgen-sensitive prostatic cancer cells, LNCaP, by dihydrotestosterone, Endocrinology 136, 796-803) and are further growth- stimulated by IGFs (Pietrzkowski. Z. , Mulholland, G. , Gomella, L. , Jameson, B. A. , Wernicke, D.. and Baserga, R. (1993) Inhibition of growth of prostatic cancer cell lines by peptide analogues of insulin-like growth factor 1 , Cancer Res. 53, 1102-1106; lwamura, M. , Sluss. P. M. , Casamento, J. B. , Cockett, A. T. (1993) Insulin-like growth factor 1 : action and receptor characterization in prostate cancer cell lines, Prostate 22, 243-252). Figure 10A confirms that androgens induce PSA expression in these cells, and that this PSA induction appears temporally related to reduction of extracellular IGFBP3 (BP3) levels as measured by ligand blotting of conditioned media (lower panel). Cell treatment with recombinant IGFBP3 selectively antagonizes androgen-dependent growth (Fig. 10B, upper panel), as does treatment with the in vitro PSA antagonist dichloroisocoumarin (DCI; Fig. 10B, lower panel). Discussion Bone density is regulated by dozens of bioactive molecules (Mohan, S. and Baylink, D. J. (1991) Bone growth factors, Clin Orthop Rel Res. 263, 30-48; Stein, G. S. , Lian. J. B. , Stein, J. L. , van Wijnen, A. J. , and Montecino, M. (1996) Transcriptional control of osteoblast growth and differentiation, Physiol Rev. 76, 593-629), many of which also affect prostate cancer cell growth. At first glance this complexity may seem to preclude clear identification of those molecules responsible for the characteristic phenotype of prostate cancer bone metastases. However, given that androgen-dependent PSA expression and osteosclerotic metastases are arguably the two most distinctive features of this malignancy, there may be a causal link between the two. Moreover, since IGFs are the most abundant class of stromal growth factors within bone (Mohan, S . and Baylink, D. J. (1991) Bone growth factors, Clin Orthop Rel Res. 263, 30-48; Rosen, C. J.. Donahue, L. R. , and Hunter, S. J. (1994) Insulin-like growth factors and bone: the osteoporosis connection. Proc Soc Exp Biol Med. 206. 83-102), androgen inducible alterations of intralesional IGFBP3 availability would seem to me to be an attractive unifying explanation for the pathogenesis of mmor osteosclerosis. The correlative nature of my data precludes definitive conclusions as to the mechanisms involved in this process. For example, it is clear from the clinical observations summarized in the Table that tumor PSA secretion is neither strictly necessary nor sufficient for the development of sclerotic lesions, thus implying the potential contribution of other 'osteoblastic' molecules capable of mimicking or enhancing the effects of PSA. A strong candidate of this type is the potent protease urokinase (uroplasmmogen activator) which, like PSA, cleaves IGFBP3 (Angelloz- Nicoud, P. and Binoux, M. (1995) Autocrine regulation of cell proliferation by the insulin-like growth factor (IGF) and IGF binding protein-3 protease system in a human prostate carcinoma cell line (PC-3), Endocrinology 136, 5485-5492) and makes IGFs more bioavailable (Koutsilieris, M. , Frenette, G. , Lazure, C , Lehoux, J. G. , Govindan, M. V. , and Polychronakos, C. (1993) Urokinase-type plasminogen activator: a paracrine factor regulating the bioavailability of insulin-like growth factors in PA-III cell-induced osteoblastic metastases. Anticancer Res. 13, 481-486) while also enhancing bone metastasis of prostate cancer cells in animal models (Achbarou, A. , Kaiser, S. , and Tremblay, G. , et al (1994) Urokinase overproduction results in increased skeletal metastasis by prostate cancer cells in vivo, Cancer Res. 54, 2372-2377). Moreover, PSA can activate urokinase either by proteolysis (Yoshida, E. , Ohmura, S. , Sugiki, M. , Maruyama, M. , Mihara, H. (1995) Prostate-specific antigen activates single-chain urokinase-type plasminogen activator, bit J Cancer 63, 863-865) or else by degrading inhibitory proteins (Espana. F. , Sanchez-Cuenca, J. , Vera, C. D. , Estelles, A. , and Gilabert, J. A (1993) Quantitative ELISA for the measurement of complexes of prostate-specific antigen with protein C inhibitor when using a purified standard, J Clin Lab Med. 122, 711-719), consistent with the existence of an androgen- dependent amplifying cascade of IGFBP3 proteases. On the other hand, the fact that urokinase expression is neither androgen-dependent nor restricted to osteosclerotic neoplasms (Andreasen, P. A. , Kjoller, L. , Christensen, L. , and Duffy, M. J. (1997) The urokinase-type plasminogen activator system in cancer metastasis: a review, Int J Cancer 72, 1-22) reduces the plausibility of this molecule as an independent trigger of osteosclerosis in prostate cancer. The two enzymes might therefore work in concert as synergistic IGFBP3 proteases - raising the possibility that rare PSA-expressing 'osteolytic' cancers could be deficient in urokinase whereas PSA-negative osteoblastic metastases may signify marked urokinase overproduction (the aggressive hormone-resistant small cell prostate cancer subtype could also explain the latter presentation, but accounts for only a minority of PSA-negative advanced cases; (Cohen, R. J. , Haffejee, Z. , Steele, G. S. , and Nayler, S. J. (1994) Advanced prostate cancer with normal PSA values, Arch Pathol Lab Med. 118, 1123-1126). Tumor-associated enzymes such as cathepsins (Conover, C. A. , Perry, J. E. , and Tindall, D. J. (1995) Endogenous cathepsin D- mediated hydrolysis of insulin-like growth factor binding proteins in cultured human prostate carcinoma cells, J Clin Endoc Metab. 80, 987- 993) or metallopro teases (Fowlkes, J. L. , Enghild, J. J. , Suzuki, K. , and Nagase, H. (1994) Matrix metalloproteinases degrade insulin-like growth factor-binding protein-3 in dermal fibroblast cultures, J Biol Chem. 269, 25742-25746) may also degrade IGFBP3 but. like urokinase, are not specifically associated with androgen-dependent tumors. Conversely, PSA-like proteases may contribute to osteosclerosis via pathways other than IGFBP3 degradation, with the apparent PSA-inducible activation of the osteoblast-stimulatory cytokine TGFβ (Killian, C. S.. Corral, D. A. , Kawinski, E. , and Constantine, R. I. (1993) Mitogenic response of osteoblast cells to PSA suggests an activation of latent TGF-β and a proteolytic modulation of cell adhesion receptors, Biochem Biophys Res Comm. 192, 940-947) not excluded by our negative data. Finally, given its 53 % homology to epidermal growth factor-binding protein (Watt, K. W. , Lee, P. J. , M'Timkulu, T. , Chan, W. P. , and Loor, R. (1986) Human prostate-specific antigen: strucmral and functional similarity with serine proteases, Proc Natl Acad Sci USA 83, 3166-3170), it is conceivable that PSA promotes a net osteoblastic reaction by sequestering this osteoclast-activating cytokine (Farsoudi, K. H. , Pietschmann, P. , Cross, H. S. , and Peterlik, M. (1993) Suramin is a potent inliibitor of calcemic hormone- and growth factor-induced bone resorption in vivo, J Pharmacol Exp Ther. 264, 579-583). The clinical phenomenon of osteoblastic metastases strongly suggests to me the existence of a classical paracrine growth loop. Given the abundance of IGF2 and IGFBP3 in normal bone (Mohan, S. and Baylink, D. J. (1991) Bone growth factors, Clin Orthop Rel Res. 263, 30-48; Rosen, C. J. , Donahue, L. R. , and Hunter, S. J. (1994) Insulin-like growth factors and bone: the osteoporosis connection, Proc Soc Exp Biol Med. 206, 83-102) - as well as the growing implication of IGF2 in both autocrine and paracrine prostate cancer growth (Figueroa, J. A. , Lee, A. V. , Jackson, J. G. , and Yee, D. (1995) Proliferation of human prostate cancer cells is inhibited by IGFBP-1 : evidence for an IGF-II autocrine growth loop. J Clin Endoc Metab. 80, 3476-3482; Angelloz-Nicoud, P. and Binoux, M. (1995) Autocrine regulation of cell proliferation by the insulin-like growth factor (IGF) and IGF binding protein-3 protease system in a human prostate carcinoma cell line (PC-3), Endocrinology 136, 5485-5492; Tennant, M. K. , Thrasher, J. B. , Twomey, P. A. , Drivdahl, R. H. , Birnbaum, R. S. , and Plymate, S. R. (1996) Protein and mRNA for type 1 insulinTike growth factor receptor is decreased and IGF2 mRNA increased in human prostate carcinoma compared to benign prostate epithelium, J Clin Endoc Metab. 81 , 3774-3782) - protease- dependent paracrine and/or autocrine positive feedback loops seem plausible, and are consistent with the additional 'autocrine' implications of my in vitro data (Fig. 10B). Of note, however, the IGF-inhibitory effects of IGFBP3 are not invariable (Narusawa, K. , Nakamura, T. , and Suzuki, K. , et al (1995) The effects of recombinant human IGF-1 and IGFBP-3 administration on rat osteopenia induced by ovariectomy and concomitant bilateral sciatic neurectomy, J Bone Mineral Res. 10, 1853-1864), suggesting that in some cell systems IGFs may be 'presented' to cells, rather than sequestered, by such binding proteins (Ernst, M. and Rodan, G. A. (1990) Increased activity of insulin-like growth factor (IGF) in osteoblastic cells in the presence of growth hormone (GH): positive correlation with the presence of the GH-induced IGF-binding protein BP- 3, Endocrinology 111, 807-814). Indeed, IGFBP3-dependent growth inhibition is not necessarily IGF-dependent (Oh, Y. , Mϋller, H. L. , Ng, L. , and Rosenfeld, R. G. (1995) Transforming growth factor-β-induced cell growth inhibition in human breast cancer cells is mediated through insulin-like growth factor-binding protein-3 action, J Biol Chem. 270, 13589-13592), perhaps indicating the existence of functional IGFBP3 receptors (Rajah, R. , Valentinis, B. , and Cohen, P. (1997) IGFBP3 induces apoptosis and mediates the effect of TGF-beta on programmed cell death through a p53- and IGF-independent mechanism, J Biol Chem. 272. 12181-12188) or else the bioactive nature of IGFBP3 degradation products (Zadeh, S. M. and Binoux, M. (1997) The 16-kDa proteolytic fragment of IGFBP-3 inhibits the mitogenic action of FGF on mouse fibroblasts with a targeted disruption of the type 1 IGF receptor gene. Endocrinology 138, 3069-3072). My clinical findings of an association between patient subgroups defined by serum PSA levels and bone lesion density require critical consideration. Like others (Oommen, R. , Geethanjali. F. S.. and Gopalakrishnan, G. , et al (1994) Correlation of serum PSA levels and bone scintigraphy in carcinoma prostate, Br J Radiol. 67, 469-471 ; Andriole, G. L. and Catalona, W. J. (1992) PSA and bone scans (letter), J IJrol. 147, 474), we have encountered occasional cases of 'PSA- negative' ( < 10 ng/ml plasma levels) bone metastases; the relative rarity of this presentation (~ 1 % ; refs. 42.52) can be gauged by published recommendations that such PSA values in effect obviate the need for isotope bone scanning in primary disease (Oesterling, J. E. , Martin, S. K. , Bergstralh, E. J. , and Lowe, F. C. (1993) The use of PSA in staging patients with newly diagnosed prostate cancer, JAMA 269, 57-60). Also like others (Soloway, M. S. , Hardeman, S. W. , Hickey, D.. Raymond. J. , Todd, B. , and Moinuddin, M. (1992) Stratification of patients with metastatic prostate cancer based on extent of disease on initial bone scan, Cancer 62, 195-202), I have failed to demonstrate any significant linear correlation between serum PSA levels and the degree of osteoblastic response in patients with PSA-positive metastatic disease. Instead, our data raise the possibility that PSA expression in metastatic tumors bears an 'all-or-none' relationship to lesional osteosclerosis, suggesting in turn that elevated serum PSA levels may not vary as a simple function of tumor PSA synthesis. This is consistent with the study of Partin et al which showed that the efficiency of mmor PSA synthesis declines with Gleason grade when corrected for disease bulk; they concluded that variations in serum PSA reflect differences in mmor 'leakiness' rather than enzyme synthesis (Partin, A. W. , Carter, H. B. , and Chan, D. W. , et al (1990) Prostate specific antigen in the staging of localized prostate cancer: influence of mmor differentiation, mmor volume and benign hyperplasia, J Urol. 143, 747-752). PSA has long been exploited as a sensitive and specific marker of metastatic prostate cancer spread. A tantalizing feature of the present study, however, relates to its implication of PSA-like proteases as potential drug targets for the palliative management of hormone-relapsed prostate cancer. Moreover, protease-dependent growth factor sensitization may prove to be a common progression mechanism in other mmor types and growth factor-dependent diseases, suggesting additional drug development opportunities. Conversely, drugs activating protease- dependent degradation of IGFBP3 could prove useful in the prevention of osteoporosis. I submit that drugs modulating the activity of proteases to growth factor-binding proteins represent an exciting new strategy for the therapeutic manipulation of growth factor receptor signalling pathways. Thus, I have obtained clinical data demonstrating a highly significant association between PSA expression and the osteoblastic phenotype of prostate cancer metastases (Fig 8 and Table 1). In addition, as part of control experiments, I have confirmed the findings of others that PSA cleaves IGFBP3 in vitro (Fig 9a) and that the amount of intact IGFBP3 may be reduced (and that of IGFBP2 increased) in the serum of patients with high PSA (Fig 9b). I have also demonstrated a selective absence of IGFBP3 in skeletal metastases of patients with osteoblastic prostate cancer when compared with osteolytic breast cancer (Fig 9c). Figures 8 and 9c represent proof of the clinical significance of PSA as a molecule responsible for the paracrine interaction of prostate cancer cells with normal bone osteoblasts in vivo, whether acting alone or by participating in a proteolytic cascade terminating in IGFBP3 proteolysis [Yoshida E et al (1995) Prostate-specific antigen activates single-chain urokinase-type plasminogen activator Int J Cancer 63, 863-865]. Control experiments confirm that LNCaP cell treatment with the synthetic androgen R1881 (R) induces a time-dependent increase in PSA expression (Fig 10a) and that a corresponding reduction of IGFBP3 concentration in the supernatant can be measured. The addition of IGFBP3 to the supernatant of androgen-treated cells antagonizes the growth-stimulatory effects of androgens (Fig 10b), consistent with a causal role for PSA- dependent IGFBP3 proteolysis in cell growth regulation. Moreover, the known PSA inhibitor dichlorisocoumarin (DCI) [Harper et al (1985) Reaction of serine proteases with substituted isocoumarins: discovery of 3,4-dichlorisocoumarin, a new general mechanism based serine protease inliibitor Biochemistry 24, 1831-1841] stimulates the antimitogenic effect of IGFBP3 (Fig 6), further supporting a growth-stimulatory effect of PSA- dependent IGFBP3 proteolysis. Addition of PSA to the supernatant is associated with only a marginal growth-promoting effect (Fig 11), but we note that for cost reasons the concentration used is only 1/1000 that mentioned by Cohen et al in reference to their aforementioned unpublished data [Cohen et al (1992) Prostate-specific antigen (PSA) is an insulin-like growth factor binding protein-3 protease found in seminal plasma J Clin Endoc Metab 75, 1046-1053] . Bone density increases, both within prostatic bone metastases and in peripheral normal bone, in tandem with the action of an IGFBP-3 protease. This increase quantitatively parallels mmor expression of the IGFBP-3 protease PSA. This is illustrated by Figs 1 to 3 of the drawings. Fig 2 shows (A) levels of PSA and (B) levels of SAP (serum alkaline phosphatase - a marker of blastic activity) for cases of prostate bone metastases. As can be seen, increased PSA is generally accompanied by increased blastic activity. Fig 3 shows: (A) a plot of SAP against PSA, illustrating the general correlation; (B) a plot of SAP against TBD (tibial - normal, peripheral, uninvolved - bone density, measured by DXA); (C) a plot of log PSA against BI (blastic index - representing the ratio of three bone densitometry measurements in diseased bone vs similar measurements in uninvolved bone from the same patient). Again, these indicate the general relationship between bone density and PSA expression. (b) Increases in bone density within metastatic mmor deposits correlate with intralesional proteolysis of IGFBP-3 (an in vitro substrate of PSA) but not with absolute expression levels of bone-regulatory growth factors Fig 4A shows a gel of IGFBP-3 protein levels in the presence of PSA, lane 1 being in the absence of PSA, while lanes 3-5 being in the presence of increasing levels of SP (seminal plasma - rich in PSA). Since PSA cleaves IGFBP-3, the level of IGFBP-3 shows the expected decrease as the level of PSA increases. Fig 4B shows a gel indicating the presence of IGFBP-3 in normal serum (lane 1) and serum from a prostate cancer patient (lane 2). Together, these observations are consistent with published results. Fig 5 shows gels indicating (A) the presence of IGFBP-3 protein, and (B) the total protein, for a series of cases, where C represents a control (recombinant IGFBP-3), " BC represents two cases of breast (lytic) metastases which are PSA-negative on immunocytochemistry, PC represents two cases of prostate metastases which are PSA-positive, and BM represents normal (control) bone marrow. Fig 5C shows (BC) a negative PSA stain in bony metastases from a breast cancer patient, and (PC) a positive PSA stain in bony metastases from a prostate cancer patient. Fig 6A shows levels of various bone-regulatory growth factors for PC and BC cases. Fig 6B shows (PSA) a validating measurement by ELISA to confirm for the PC cases that the bone samples did indeed contain prostatic factor, and (CK) an antibody blot measurement for the BC cases of cytokeratins, which are accepted as evidence of epithelial-derived carcinoma within a mesenchymal (eg marrow) sample. Whereas Fig 5 indicates a significant lowering of IGFBP-3 in prostate cancer metastases as compared with breast cancer metastases, no such difference is seen in Fig 6 for the various bone-regulatory growth factors FGF-2, TGF-β 1 , IGF-1 , TGF-α and EGF. Androgen-stimulated LNCaP cells are well known to be growth-stimulated by IGF-I 42 ' 41 . Dichloroisocoumarin (DCI) is a broad-spectrum serine protease inhibitor 44 known to inhibit PSA 45 . This reagent increases IGFBP medium of LNCaP cells, and inhibits their androgen-dependent growth (data not shown here). Taking as precedent the observation that a bradykinin inliibitor cross-inhibits PSA 45 , we used a panel of commercially available inhibitors of angiotensin-converting enzyme (ACE), which, like bradykinin, is a serine protease of the kallikrein family, to inhibit PSA and/or other IGFBP-3 proteases. The inhibitor lisinopril was the most potent of those tested. Fig 7A shows a gel of PSA protein produced over a number of days in LNCaP cells stimulated with androgen (R1881 - the synthetic androgen metribolone); lane 1 being at time zero, while lanes 2-4 are at days 1 , 2 and 3 respectively. Fig 7B shows the growth over time of LNCaP cells in the presence of androgen (R1881) compared with a control. As expected, the level of PSA and cell growth is higher with androgen stimulation. Fig 7C shows the effect of BP3 (recombinant IGFBP-3) on cell growth, where C is the untreated control, and R indicates the presence of androgen (R1881). It is clear from this that IGFBP-3 reduces cell growth, particularly where it has been stimulated by androgen. Fig 7D is a gel showing the effect of DCI (a known PSA inhibitor) on IGFBP-3. Lane 1 is a positive control, containing just recombinant IGFBP-3. Lane 2 is control medium, showing minimal amounts of IGFBP-3, while lane 3 is medium in the presence of DCI, showing an increased amount of IGFBP-3 compared with lane 2. Fig 7E shows the effect of DCI on the androgen-dependent cell growth. C is the control, with no androgen and no DCI. R indicates stimulation with androgen (R1881). The growth was measured after 3 days. It clearly shows that PSA inhibition, like addition of IGFBP-3, reduces androgen- stimulated cell growth. Fig 7F shows a gel of IGFBP-3 levels in the presence of the protease inhibitor lisinopril, where lane 1 is untreated cell medium (ie no protease inliibitor) at time t=0, lanes 2 and 3 are the same at t=24 hr and act as controls for the lanes 4-6 which are also measured at 24 hr, lane 4 shows the effect of androgen stimulation (lowered IGFBP-3 resulting from increased PSA), lane 5 shows increased IGFBP-3 in the presence of the protease inhibitor lisinopril, lane 6 shows the effect of combined androgen and lisonopril. Fig 7G shows the effect of linisopril on cell growth (after 3 days). C indicates the untreated control cells, LIS indicates the presence of lisinopril, and R indicates the presence of androgen (R1881). The results show that linisopril, like IGFBP-3 and DCI, reduces androgen-stimulated cell growth. Thus, the results indicate that lisinopril, the most potent of three drugs tested - the others being captopril and enalapril; data not shown - mimics the action of DCI in upregulating IGFBP-3 expression and inhibiting androgen-dependent LNCaP cell growth. X Example 2: Identification of candidate BPP inhibitors using in vitro drug effects on GFBPs as an endpoint A method to identify candidate drugs for the treatment of growth factor- dependent diseases involves using existing knowledge of a relevant interaction between a GFBP and a growth factor-binding protein protease (BPP) which is known to cleave or destroy said GFBP: BPP protease → BPP → GFBP → GF → GFR In this assay it is desirable to know the identity and sequence of the relevant GFBP and BPP, or have purified or recombinant proteins. In the simplest example of this approach, recombinant or purified BPP and GFBP (or BPP protease and BPP) are obtained either from (1) commercial or collaborative sources, or (2) by obtaining sequence data using available computer databases [Fickett JW (1996) "Finding genes by computer; the state of the art" Trends Genet (1996) 12, 316-320] or publications (eg for the BPP, PSA [Watt et al (1986) "Human prostate-specific antigen: strucmral and functional similarity with serine proteases" Proc Natl Acad Sci USA 83, 3166-3170; Lundwall A (1989) "Characterization of the gene for prostate-specific antigen, a human glandular kallikrein" Biochem Biophys Res Comm 161 , 1151-1159; Digby et al (1989) "Human prostate- specific antigen gene: strucmre and linkage to the kallikrein-like gene hGK-1 " Nucl Acids Res 17, 2137; Henttu P & Vihko P (1989) "cDNA coding for the entire human prostate specific antigen shows high homologies to the human tissue kallikrein genes" Biochem Biophys Res Comm 160, 903-910; Schulz et al (1988) "Sequence of a cDNA clone encompassing the complete mature human prostate specific antigen (PSA) and an unspliced leader sequence" Nucl Acids Res 16, 6226] and its GFBP substrate, IGFBP3 [Spratt et al (1990) "Cloning and expression of human insulin-like growth factor binding protein 3" Growth Factors 3, 63-72; Albiston et al (1990) "Cloning and characterization of the growth hormone-dependent IGFBP-3 in the rat" Biochem Biophys Res Comm 166, 892-897]), or by obtaining cDNAs from commercial libraries (eg OriGene Technologies). Using standard procedures the PCR-derived cDNAs can then be packaged in an appropriate expression system, used to produce the recombinant protein (eg in baculovirus-infected insect cells) of interest [Karr et al (1995) "The presence of PSA-related genes in primates and the expression of recombinant PSA in a transfected murine cell line" Cancer Res 55, 2455-62] , and purified as described [Lovgren et al (1995) "Production of recombinant PSA and HK2 and analysis of their immunologic cross-reactivity" Biochem Biophys Res Comm 213, 888- 895] . With the two proteins in hand, the protein detection system in use (eg ELISA or Western blot) is used to determine a minimum or otherwise appropriate amount of solubilised GFBP to include in the assay for easy detection. Having determined from relevant published sources the appropriate buffer and temperamre for maximising the enzyme activity of the BPP, separate aliquots of the GFBP control are mixed with increasing concentrations of the BPP for a given time (say, 60 mins) to determine the minimum amount which must be added to induce readily detectable GFBP proteolysis, as detected either by a reduction in the signal intensity or by the appearance of degraded fragments [Lamson et al (1991) "A simple assay for proteolysis of IGFBP-3" J Clin Endoc Metab 72, 1391-1394] . At this point the assay is ready for screening candidate drugs (D) which can be added at various concentrations in the nanomolar to micromolar range, with such addition occurring either prior to or at the same time as BPP addition. If addition of a given D is associated with increased GFBP signal or reduction of GFBP degradation, a candidate BPP inhibitor has been identified. Such a compound D can then be incorporated into further assays (eg cell growth, xenograft growth) to evaluate its suitability for clinical testing. The assay can be readily converted to high-flux screening using established protocols (Harding et al (1997) "Development of an automated high-throughput screening system" Drug Discovery Today 2, 385-391), and a library of suitable compounds for testing can be produced using combinatorial chemistry (Haiduk et al (1997) "Discovering high- affinity ligands for proteins" Science 278, 497-499). Example 3: Candidate drug identification using in vitro binding to GFBPs as an endpoint It is desirable to know the identity of the GFBP and its size, but it is not necessary to know the identity of the GFBP protease or other upstream regulators. This approach is particularly useful for identifying drugs which interfere with the binding of GFs to GFBPs, hence increasing the net activity of the GF in question. To identify candidate small-molecule drugs for enhancing the activity of a given GF, an appropriate amount of purified or recombinant GFBP is electrophoresed and transferred to a membrane. Strips of the membrane are then exposed to candidate drugs at a range of concentrations for (say) 30-60 mins, then washed and re-incubated with radioligand. Figure 14 shows how this approach can be used to visualize " ^I-EGF binding (for a fuller description of this technique, see Komatsu et al (1996) "Epidermal growth factor binding proteins in rice" Biol Pharm Bull 19, 905-908). A reduction in the signal identifies a candidate drug. This version of the assay can also be adapted to ELISA for high- throughput screening, in which case iodination of the ligand will be unnecessary. It can also be used on cell lysates or can be adapted to column chromatography or immunoprecipitation. Example 4: Candidate drug identification using cell-based GFBP effects as an endpoint A suitable cell line known to express GFBP is required. In this approach, a GFBP-expressing cell line is treated with a panel of candidate drugs and controls, for a range of times (up to, say, 24h) then lyzed. Each of the lysates are incubated with radioligand, then immunoprecipitated with a GFBP antibody. The immunoprecipitates are washed, then boiled, and an aliquot examined by scintillation counting. Samples in which there is a significant increase or decrease in radioactivity identify candidate drugs for further evaluation in binding and biological assays. For high-throughput screening, the immunoprecipitation step can be avoided by creating an ELISA assay. In this version, candidate drugs are used to treat the living cells (or else applied directly to the lysate, though this is only likely to identify GFBP-blocking drugs). The ELISA plate is coated with GFBP antibody, and the cell lysates are then applied to the wells. Following washing of the plate, ligand (which may be unlabelled) is applied for an appropriate period (say 30 mills), then washed. Ligand antibody is then applied, incubated, subsequently washed, and the binding quantified using standard procedures. Again, any significant increase or decrease in binding identifies candidate drugs for further character izaton. The advantage of this approach is that the details of the biology are not essential, and drugs affecting any part of the pathway will be identified. Example 5: Treatment of prostate cancer with an angiotensin converting enzyme (ACE) inhibitor, lisinopril The method of the invention has been used to identify a candidate drug for the treatment of prostate cancer. Given the known mutual involvement of kallikrein-like serine proteases (which include PSA) and the metallopeptidase angiotensin-converting enzyme (ACE) in the metabolism of kinin proteins, we screened a panel of ACE inhibitors for PSA- inhibitory activity based on IGFBP3 expression and integrity in vitro and in vivo. Control experiments showed that treatment of androgen- stimulated LNCaP cells with captopril, enalapril or lisinopril enhanced IGFBP3 levels; the greatest effect was seen with lisinopril (Fig. 12a). Similarly, in vitro co-incubation of PSA and lisinopril mimicked the PSA- inhibiting effects of DCI on IGFBP3 proteolysis (Fig. 12b). Having thus implicated lisinopril as a candidate PSA inhibitor, this drug was used to treat androgen-stimulated LNCaP cells in a cell growth assay; Figure 13 shows that the effect of lisinopril in this assay is indeed growth-inhibitory. In this respect I note also the isolated observation of a hypertensive patient with metastatic osteosclerotic prostate cancer resistant to hormone manipulation, who, in the absence of any recent change in antiandrogen treatment, or prescription of cytotoxic therapy or radiotherapy, was commenced on captopril. His elevated serum alkaline phosphatase, a marker of osteoblastic response, declined by more than 50% (from — 900 to —400) over the next month. To the surprise of his doctors, he remained reasonably well for a period of some further months. These findings illustrate the feasibility of using GFBP-based assays for the identification of candidate drugs, and provisionally implicate certain ACE inliibitors as useful palliative drugs in PSA-expressing prostate cancer. Example 6: New treatment of osteoporosis To develop a new treatment of osteoporosis, a drug is sought to mimic or augment the activity of the pro-osteoblastic cytokine IGF-1. For this task, the methods in the previous Examples are used to identify candidate drugs. For the method as described in Example 3, IGFBP3 is purchased (Sigma) and dissolved in phosphate-buffered saline (PBS) at a concentration of 100 ng/ml. A test run confirms that electrophoresis of 50 μl of this solution is readily detectable by I-IGF radioligand blotting. An identical series of such samples is then electrophoresed, transferred, and separately exposed to candidate drugs for 30 mins at room temperamre on a rolling platform. After washing, the membrane strips are re-exposed to radioligand, with any 'negative' samples being judged 'positive' in the assay. For the method as described in Example 4, a series of cell samples are treated with candidate drugs (including dummy treatments). The samples are then lyzed, and the lyzates are then mixed with I-IGF for 60 mins at 4°C on a rotator. A commercially available anti-IGFBP3 antibody is then added to each sample at a 1 : 100 dilution, and mixed for a further 30 mins. 40μl of Protein A sepharose beads (Promega) are then added to the sample as a slurry, mixed for a further 30 mins, then gently centrifuged to pellet the beads which are washed thrice in PBS. The washed beads are then resuspended in boiling buffer, boiled, and an aliquot submitted for scintillation counting. Those samples which measure significantly lower than dummy-treated samples identify candidate drugs for further rounds of screening and biological testing. Example 7: New drugs for scleroderma A screening method is described for developing new drugs for treating scleroderma. Knowing that excessive TIMP-1 activity has been linked to the low collagenase levels in this disease, candidate drugs are screened using fibroblast production of the PDGF-binding extracellular matrix protein, collagen IV, as an in vitro endpoint. It is immaterial whether the drugs inhibit TIMP-1 , potentiate collagenase, or inhibit the activity of PDGF by altering its interaction with collagen IV, the assay described in Example 4 is used but includes an assay as described in Example 3 as a backup. For the assay of the type described in Example 4, MRC-5 human fibroblasts are treated with a variety of drugs, including dummy compounds, for periods ranging up to 72 h. The cell samples are then lyzed. Antibodies to collagen IV are sought commercially, but if unavailable are produced on a custom basis using synthetic peptides based on published sequence information. These antibodies are then used to coat wells for an ELISA assay. Drugs inducing a significant decrease in the collagen IV signal are identified as candidate compounds for further characterization. For the assay of the type described in Example 3, the initial assumption that collagen IV potentiates PDGF action by 'presenting' it to fibroblasts (ie rather than sequestering it) is made and compounds which displace PDGF from collagen are identified. Recombinant PDGF-BB is purchased from commercial sources and radioiodinated in house; the published collagen IV sequence is used to design PCR primers by which the cDNA is obtained from an MRC-5 library, and this is in turn used to synthesise recombinant collagen IV from a baculovirus/insect cell expression system. 96-well plates are then coated with the recombinant collagen IV, and a panel of compounds applied to the plates. Following washing, a small amount (eg picomolar) 19c of I-PDGF is added to each well for 15 mins at room temperamre, then removed and each supernatant subject to scintillation counting. A higlier- than-control reading identifies compounds which prevent radioligand binding to the GFBP, though abnormally low readings also identify compounds of future interest. This approach requires careful initial standardization, but once developed can be readily used for high-flux screening, and is easily adapted to other growth factors (eg TGF-β). Example 8: New drugs for male-pattern baldness A drug for treating autosomal dominant male-pattern baldness is sought. It is hypothesised that the underlying defect in this disorder relates to deficient activation of the precursor molecule for epidermal growth factor (pro-EGF; the GFBP of this example) by the kallikrein-like serine protease EGF-binding protein (EGFBP). Using the standard methodologies alluded to in the earlier examples, recombinant pro-EGF and EGFBP is produced which is then incorporated into the following variation of the assay of Example 2. 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Claims: CLAIMS 1. A method of identifying or developing a compound which is useful in treating a growth factor-dependent disease, the method comprising the step of determining whether the compound modulates the amount, function or strucmral integrity of a growth factor binding protein. 2. A method according to Claim 1 wherein the compound modulates the activity of a protease, which protease is capable of modulating the amount, function or strucmral integrity of the growth factor binding protein. 3. A method according to Claim 1 wherein the compound modulates the binding of the growth factor to the growth factor binding protein. 4. A method according to Claim 1 wherein the disease is one involving known or suspected protease dysregulation. 5. A method according to Claim 2 wherein the protease is capable of proteolytically degrading the growth factor binding protein. 6. A method according to Claims 2, 4 or 5 wherein the compound increases the activity of the protease. 7. A method according to any one of Claims 2, 4 or 5 wherein the compound decreases the activity of the protease. 8. A method according to any one of Claims 2 or 4 to 7 wherein the compound directly modulates the activity of the protease. 9. A method according to any one of Claims 2 or 4 to 8 wherein the compound indirectly modulates the activity of the protease. 10. A method according to Claim 7 wherein the compound inhibits protease activity. 11. A method according to any one of the Claims 2 or 4 to 10 comprising the steps of providing a growth factor binding protein and a protease which is capable of proteolytically-degrading the binding protein, and determining whether the compound modulates the proteolysis of the binding protein by the protease. 12. A method according to any one of the preceding claims wherein the growth factor binding protein is any one of a binding protein which binds to a growth factor selected from any of polypeptide growth factors which are capable of binding transmembrane kinases; cytokines which are capable of binding hetero-oligomeric non-catalytic cytokine receptors; peptide hormones which are capable of binding seven-transmembrane- domain G-protein coupled receptors; and neurotransmitters which are capable of binding ligand-gated ion channels. 13. A method according to any one of the preceding claims wherein the growth factor binding protein is any one of a binding protein which binds epidermal growth factor, transforming growth factor beta, erythropoietin, growth hormone, an interleukin, an interferon, platelet-derived growth factor BB, basic fibroblast growth factor, and insulin-like growth factors I and II. 14. A method according to Claim 13 wherein the growth factor binding protein is insulin-like growth factor binding protein-3. 15. A method according to Claim 12, when dependent on any of Claims 2 or 4 to 10 wherein the protease is prostate specific antigen. 16. A method of treating a growth factor-dependent disease, the method comprising the steps of administering to the patient an effective amount of a compound which modulates the amount, function or strucmral integrity of a growth factor binding protein. 17. A method according to Claim 16 wherein the compound modulates the activity of a protease which protease is capable of modulating the amount, function or strucmral integrity of the growth factor binding protein. 18. A method according to Claim 16 or 17 wherein the disease is one involving known or suspected protease dysregulation. 19. A method according to Claims 17 or 18 wherein the compound increases the activity of the protease. 20. A method according to any one of Claims 17 or 18 wherein the compound decreases the activity of the protease. 21. A method according to any one of Claims 17 to 20 wherein the compound directly modulates the activity of the protease. 22. A method according to any one of Claims 17 to 20 wherein the compound indirectly modulates the activity of the protease. 23. A method according to Claim 21 wherein the compound inhibits protease activity. 24. A method according to any one of Claims 16 to 23 wherein the growth factor binding protein is any one of a binding protein which binds to a growth factor selected from any of polypeptide growth factors which are capable of binding transmembrane kinases; cytokines which are capable of binding hetero-oligomeric non-catalytic cytokine receptors; peptide hormones which are capable of binding seven-transmembrane- domain G-protein coupled receptors; and neurotransmitters which are capable of binding ligand-gated ion channels. 25. A method according to any one of Claims 16 to 24 wherein the growth factor binding protein is any one of a binding protein which binds epidermal growth factor, transforming growth factor beta, erythropoietin, growth hormone, an interleukin, an interferon, platelet-derived growth factor BB, basic fibroblast growth factor, and insulin-like growth factors I and II. 26. A method according to Claim 22 when dependent on Claim 17 wherein the protease is capable of inactivating insulin-like growth factor binding protein-3. 27. A method according to Claim 26 wherein the protease is prostate specific antigen. 28. A method of treating prostate cancer, the method comprising administering to the patient an effective amount of a compound that modulates the amount, function or structural integrity of insulin-like growth factor binding protein-3. 29. A method of treating prostate cancer, the method comprising administering to the patient an effective amount of a compound that reduces the activity of a protease which protease is capable of inactivating a insulin-like growth factor binding protein-3. 30. A method according to Claim 29 wherein the protease is prostate- specific antigen. 31 . A method of treating osteopaenic bone disease such as osteoporosis the method comprising administering to the patient an effective amount of a compound that modulates the amount, function or strucmral integrity of insulin-like growth factor binding protein-3. 32. A method of treating osteopaenic bone disease such as osteoporosis, the method comprising administering to the patient an effective amount of a compound that increases the activity of a protease which protease is capable of inactivating a insulin-like growth factor binding protein-3. 33. A method of treating prostate cancer, the method comprising administering to the patient an effective amount of an angiotensin- converting enzyme inhibitor. 34. A method according to Claim 33 wherein the angiotensin- converting enzyme inhibitor is any one of lisinopril. enalapril or captopril. 35. A method according to Claim 34 wherein the angiotensin- converting enzyme inhibitor is lisinopril. 36. Use of a compound which modulates the amount, function or strucmral integrity of a growth factor binding protein in the manufacmre of a medicament for treating a growth factor-dependent disease. 37. Use of a compound that modulates the amount, function or strucmral integrity of a growth factor binding protein in the manufacmre of a medicament for treating prostate cancer. 38. Use of an angiotensin-converting enzyme inhibitor in the manufacmre of a medicament for treating prostate cancer. 39. Use of a compound that modulates the amount, function or structural integrity of a growth factor binding protein in the manufacmre of a medicament for treating osteopaenic bone disease, such as osteoporosis.

Inventors: EPSTEIN RICHARD JOHN, EPSTEIN, RICHARD, JOHN
Applicants: EPSTEIN RICHARD JOHN, IMP EXPLOIT LIMITED, EPSTEIN, RICHARD, JOHN, IMPERIAL EXPLOITATION LIMITED
Application year: 1998
Main classification: G01N33
IPC codes: A61K31, A61K38, A61K45, A61P19, A61P35, A61P43, G01N33