CA2224859A1

CA2224859A1 - Ligand inhibitors of insulin-like growth factor binding proteins and methods of use therefor

Info

Publication number: CA2224859A1
Application number: CA002224859A
Authority: CA
Inventors: Amitabh Gaur; Xin-Jun Liu; Nicholas Ling; Dominic Behan
Original assignee: Individual
Current assignee: Neurocrine Biosciences Inc
Priority date: 1996-04-17
Filing date: 1997-04-17
Publication date: 1997-10-23
Also published as: EP0854884A1; MX9710291A; JPH11508608A; AU2676297A; WO1997039032A1

Abstract

Methods for increasing the level of free, biologically active proteins, including insulin-like growth factors, and for treating IGF-responsive conditions are provided. The methods generally comprise administering one or more ligand inhibitors that inhibit the binding of the protein to one or more insulin-like growth factor binding proteins. Suitable ligand inhibitors include analogs of IGF-I or IGF-II and small molecule inhibitors.

Description

CA 022248~9 1997-12-17 Wo 97139032 PCT/US97/06503 Description LIGAND INHIBITORS OF INSULIN-LIKE GROWTH FACTOR BINDlNG
PROTEINS AND METHODS OF USE THEREFOR

Technical Field The present invention relates generally to compositions and methods for the treatment of conditions responsive to the ~-lmini~tration of insulin, human growth 10 hormone, insulin-like growth factor, and/or other proteins that bind to insulin-like growth factor binding proteins. The invention is more particularly related to ligand inhibitors that inhibit the binding of insulin-like growth factor to insulin-like growth factor binding proteins, and to the use of such inhibitors for ~(lmini~tration (e.g., orally) to patients for the trç~tment of diabetes, neurodegenerative diseases and other disorders.
Back~round of the Invention Insulin-like growth factors (IGFs) are polypeptide hormones that are structurally similar to each other and to insulin. Two IGFs, known as IGF-I and IGF-II, have been identified. and both have a variety of metabolic actions and affect the growth 20 of multiple cell types (see, e.g.~ LeRoith and Roberts, "Insulin-like Growth Factors,' Ann. NYAcad Sci. 692:1-9, 1993). IGF-I is a 70 amino acid peptide with a molecular weight of 7649 and three disulfide bridges. Its actions in vivo include the mediation of growth hormone actions and bone deposition and maturation. IGF-I also mimics theaction of insulin, and the IGF-I receptor has high homology to the insulin receptor.

2~ IGF-II is strongly homologous to IGF-I and this factor plays a role in, for example, bone remodeling and brain cell m~int-~n~nce and differentiation.
While IGF-I is present in a wide variety of body tissues, it is normally found in an inactive forrn in which it is bound to an IGF binding protein (IGFBP or BP). Six related BPs are known and have been designated IGFBP1-IGFBP6 (see, e.g., 30 Holly and Martin? "Insulin-like Growth Factor Binding Proteins: A Review of Methodological Aspects of Their Purification~ Analysis and Regulation~" G)owth CA 022248~9 1997-12-17 W O 97139032 PCTrUS97/06503 /

Regul. 4(Suppl 1):20-30, 1994; Langford et al., "The Insulin-like Growth Factor-I/Binding Protein Axis: Physiology, Phytophysiology and Therapeutic Manipulation,"
Eur. J. Clin. Invest. 23(9~:503-16, 1993). BPs play an important role in IGF regulation by exerting inhibitory and/or stim~ tory effects on IGF action. For example, about 5 90% of circulating IGF-I is present in a trimolecular complex containing IGFBP-3 and acid labile subunit (ALS). The IGF-I within such complexes is unable to bind to surface receptors, and is therefore biologically inactive. IGF-I present within the trimolecular complex also has a substantially longer half-life than uncomplexed IGF-I.
Attempts have been made to treat a wide variety of diseases by 10 ~flministration of IGF-I, IGF-II or an IGF binding protein. For example, the use of IGF-I for the treatment of cardiac disorders, intestinal disorders and osteoporosis are described in U.S. Patent ~o. 5,126,324, WO 91/12018 and European Patent Application 560,723, respectively, and the use of IGF-I for enhancing growth is described in U.S. Patent No. 5,126,324. IGF-I has also been studied for use in treating 15 insulin-resistant states and diabetes (see, e.g, Clemmons and Underwood, "Uses of Human lnsulin-Like Growth ~actor-I in Clinical Conditions,".J. Cli~ Endocrinol.
Metab. 79(1):4-6, 1994; Langford et al., "The Insulin-like Growth Factor-l/Binding Protein Axis: Physiology, Phytophysiology and Therapeutic Manipulation," E~r. J.Clin.Invest. 23(9):503-16,1993). Therapiesinvolvingthe~lminictrationofantibodies20 raised against IGF-I and specific binding proteins are described, for example, in WO
94/04569 and WO 92/14834, respectively.
Like treatment with insulin or growth hormone, however, treatment with intravenous IGF or antibodies thereto generally requires repeated intravenous injection, resulting in a high cost and practical difficulties for patients. Such treatments can also ~5 induce side effects due, for example, to the inahility to target specific tissues within the body. Further, the scope of treatment is limited to the tissues that may be reached by intravenous hormone ~ministration.
Accordingly, there is a need in the art for improved efficiency and control in treating conditions responsive to IGF and/or other proteins that bind to 30 insulin-like growth factor binding proteins. The present invention fulfills these needs and further provides other related advantages.

CA 022248~9 1997-12-17 Wo 9713go32 PCTrUSg7/06~03 Summary of the Invention Briefly stated, the present invention provides therapeutic and screening methods employing ligand inhibitors that inhibit the binding of proteins such as insulin-like growth factors to insulin-like growth factor binding proteins. In one aspect, the present invention provides a method for increasing the level of free, biologically active insulin-like growth factor in a patient, comprising ~riministering to a patient one or more ligand inhibitors that inhibit the binding of an insulin-like growth factor to one or more insulin-like growth factor binding proteins, and thereby increasing the level of free~ biologically ~ctive insulin-like growth factors within the patient.
ln a related aspect, methods are provided for treating an IGF-responsive condition in a patient, comprising ~mini.stering to a patient one or more ligandinhibitors that inhibit the binding of an insulin-like growth factor to one or more insulin-like growth factor binding proteins, and thereby alleviating an IGF-responsive condition in a patient.
In another aspect, the present invention provides a pharmaceutical composition comprising: (a) one or more ligand inhibitors that inhibit the binding of an insulin-like growth factor to one or more insulin-like growth factor binding proteins;
and (b) a physiologically acceptable carrier.
In yet another aspect, the present invention provides a method for screening for a small molecule inhibitor that inhibits binding of an insulin-like growth factor to an insulin-like growth factor binding protein, comprising: (a) combining an insulin-like growth factor with an insulin-like growth factor binding protein in a solution cont~ining a candidate small molecule, such that the binding protein and the growth factor are capable of forming a complex; and (b) determining the amount of complex in the solution, relative to a predetermined level of binding in the absence of the small molecule, and therefrom evaluating the ability of the small molecule to inhibit binding of an insulin-like growth factor to an insulin-like growth factor binding protein.
In still another aspect, methods are provided for increasing the level of a free protein in a patient, comprising a~mini.stering to a patient one or more ligand CA 022248~9 1997-12-17 inhibitors that inhibit the binding of a protein to one or more insulin-like growth factor binding proteins, and thereby increasing the level of the free protein within the patient.
These and other aspects of the present invention will become al,pale-lL
upon reference to the following detailed description and attached drawings. All 5 references disclosed herein are hereby incorporated by reference in their entirety as if each was incorporated individually.

Brief Description of the Drawings Figure 1 is a graph that depicts the relative amount of binding, expressed 10 as cpm, of l25I-labeled IGF-I tracer to IGFBP-3, in the presence of varying amounts of [T59]hIGF-I, hIGF-I and [L24,59,60,A31~hIGF-I.
Figure 2 is a graph that presents the relative amount of DNA synthesis, expressed as cpm of incorporated [3H]-thymidine, of 3T3 cells in the presence ofvarying amounts of [T59]hIGF-I and [L24,59,60,A3 l]hIGF-I.
Figure 3 is a graph that depicts the relative amount of DNA synthesis, expressed as cpm of incorporated [3H]-thymidine, of 3T3 cells in the presence ofmedium only (column 1), 10 nM [T59]hIGF-I (column 2) and 10 nM [T59]hIGF-I plus 25 nM IGFBP-3 (column 3). The figure also shows the relative amount of DNA
synthesis in the presence of 10 nM [TS9]hIGF-I plus 25 nM IGFBP-3, with the addition 20 of varying amounts of [L24,59,60,A3 1 ]hIGF-I.
Figure 4 is a graph that shows the decrease in blood glucose levels (in mg/dl) over time (expressed in minutes) following ~lministration of [L24,59,60,A3 1 ]hIGF-I to diabetic NOD mice.
Figure 5 is a pair of graphs that depict the effect of insulin and 25 IGFBP3-LI ([L24,59,60,A31~hIGF-I) on plasma glucose in rats treated systemically with glucose.

Detailed Description of the Invention As noted above, the present invention is generally directed to methods 30 employing ligand inhibitors for increasing the level of a free protein, such as biologically active IGF, in one or more tissues within a patient. The proteins affected CA 022248~9 1997-12-17 W 097/39032 PCT~US97106503 by ligand inhibitors as described herein are generally proteins that bind to one or more IGF binding proteins. Increasing the level of such a protein within a patient may generally be useful in the treatment of a variety of conditions. Within the context of the present invention, "IGF" refers to one or more insulin-like growth factors (i.e., IGF-I
5 and/or IGF-II). IGFs are peptide hormones, and the sequences of IGF-I and IGF-II in humans and other species have been determined. Human IGF-I (hIGF-I) is a 70 amino acid peptide which has the sequence shown in Figure 1 (SEQ ID NO:l). "Free"
protein, such as IGF, refers to protein that is not complexed or bound to an IGF binding protein.
An "IGF binding protein" (IGFBP or BP) is any protein that binds to IGF-I and/or IGF-II in vivo, resulting in the inhibition of IGF binding to one or more cell surface receptors, or soluble forms thereof. A BP binds to IGF (i.e., forms a complex) through noncovalent interactions. IGF binding proteins contemplated within the context of the present invention include IGFBP-l, -2, -3, -4, -5 and -6. In particular, IGFBP-3, the most abundant binding protein in adult serum, has a high affinity for both IGF-I and IGF-II. Binding to IGFBP-3 increases the half life of IGF-I from about 10 minutes to approximately 15 hours (see Langford and Miell, ~20. J. Clin. Invest.23:503-526, 1993), and is therefore important for controlling the level of IGF-I in the circulation.
A '~ligand inhibitor," within the context of the present invention, is any molecule (other than an antibody to IGF-I or IGF-II) that is capable of inhibiting the binding of one or more proteins, especially IGF-I and/or IGF-II, to one or more IGFBPs. Due to the similarity between the structures of IGF-I and IGF-II, it will be apparent that many ligand inhibitors will inhibit the binding of both IGF-I and IGF-II to an IGFBP. In some cases, the binding of one IGF will be inhibited to a greater extent than that of the other IGF. In other cases, however, a ligand inhibitor may be specific for IGF-I or IGF-II. A ligand inhibitor may displace IGF from a complex with a BP?
thereby causing bound IGF to become free IGF. Such displacement may be reversible or irreversible. A ligand inhibitor may also block the binding of free IGF to a BP
because of a high affinity for either IGF or one or more BPs. For example, a ligand inhibitor may bind to IGF within a BP binding site, or may bind to a BP at an IGF

CA 022248~9 1997-12-17 Wo 97/39032 PCT/US97/06503 binding site. Alternatively, a ligand inhibitor may bind to IGF or a BP at a site that is not such a binding site, and inhibit complex formation through an allosteric interaction.
A ligand inhibitor having a lower affinity for IGF or one or more BPs may also inhibit the binding of free IGF to a BP when present at high enough concentrations. Similar 5 mech~nism.~ of inhibition may also be observed for ligand inhibitors directed against other proteins that bind to a BP.
As used herein, a molecule "inhibits" binding of a protein to an IGFBP
if the level of free protein increases by about 10-30% or more. An increase in the level of free protein may generally be detected using a variety of assays known to those of 10 ordinary skill in the art, such as im~ging, radioimmunoassays and precipitation techniques as described herein. One such assay is described in Example 1, below.Preferably, the characteristics of the ligand inhibitor are such that it has a 100 fold selectivity to the IGFBP (Kj < 10 nM).
Ligand inhibitors include, but are not limited to, analogs of IGF-I or 15 IGF-II and small molecule inhibitors. An IGF "analog" is a peptide that has an amino acid sequence that is substantially identical to a native IGF sequence, but that has one or more amino acid substitutions and/or modifications. Such substitutions and/ormodifications may reduce the biological activity of the peptide (i.e., decrease the affinity of the peptide for one or more cell surface receptors) without decreasing the 20 ability of the peptide to bind to one or more BPs. A "small molecule inhibitor" is a ligand inhibitor that is a natural or synthesized non-peptide, organic molecule. Small molecule inhibitors are typically identifled by screening libraries obtained from soil samples, plant extracts, marine microorg~ni~m~, fermentation broth, fungal broth, pharmaceutical chemical libraries, combinatorial libraries (both chemical and 25 biological) and the like. Such libraries may be obtained from a variety of sources, both commercial and proprietary.
One preferred ligand inhibitor of the present invention is the [L24,A3 1 ,L59,L60] analog of hIGF-I. This analog (which has the sequence recited in SEQ ID NO:2) binds to IGFBP-3, but not to the IGF-I cell surface receptor.
30 [L24,A31,L59,L60]hIGF-I may generally be prepared by techniques well known to those of ordinary skill in the art, such as by chemical synthesis.

CA 022248~9 1997-12-17 It will be a,~,pa~ t to those of ordinary skill in the art that modifications may be made to the sequence of [L24,A3 1 ,L59,L60]hIGF-I such that the ability of the analog to inhibit the binding of IGF-I to IGFBP-3 is retained. Such variants are within the scope of the present invention, and may generally be identified by modifying the 5 sequence and evaluating the inhibitory plo~ lies of the analog as described below.
Preferably, a variant contains conservative substitutions, (i.e., one in which an amino acid is substituted for another amino acid that has simi}ar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure andhy-llvlJ~Lllic nature of the polypeptide to be substantially unchanged). Amino acids 10 suitable for conservative substitutions include those having functionally similar side chains. For example, a hydrophobic residue (e.g., glycine, alanine, valine, leucine, isoleucine and methionine) may replace another such residue. Similarly, conservative substitutions may involve interch~nging hydrophilic residues (e.g, arginine and Iysine, glutamine and asparagine, threonine and serine), basic residues (e.g, lysine, arginine 15 and histidine), and/or acidic residues (e.g., aspartic acid and glutamic acid). Variants may also (or alternatively) be modified by, for example, the deletion or addition of amino acids, or the chemical modification of amino acids, that have minim~l influence on the inhibitory properties of the analog.
Small molecule inhibitors according to the present invention may be 20 prepared using methods well known to those of ordinary skill in the art. According to a preferred method, a chemical library of small molecules as described above may be screened using a binding assay designed to detect molecules that displace a protein such as IGF from a binding protein. For example, a complex of radiolabeled IGF-I and a binding protein (such as BP-3) may be incubated in the presence of a candidate small 25 molecule. Complexes (i.e., noncovalent associations of IGF and binding protein) may then be separated from the rem~in-ler of the solution using, for example, polyethylene glycol precipitation. Those small molecules that bind to the complex and displace the growth fàctor from the binding protein may then be detected by a decrease in theamount of radiolabel precipitated. Those of ordinary skill in the art will recognize that 30 a variety of other assay formats may be employed in such a screen, including two-site sandwich ELISAs, chemiluminescent assays and fluorescent assays.

.. .

CA 022248~9 1997-12-17 W O 97~9032 PCT~US97/06503 IGF-I, IGF-II and binding proteins for use in such assays may generally be prepared by techniques known to those of ~rdill~ y skill in the art, such as those provided in Rechler, ~itamins and Hormones 4'7:1-114, 1993, and references citedtherein. Appropriate techniques include chemical synthesis (described, for example, in 5 Shim~ki et al., J. Biol. C~em. 266:10646-10653, 1991), purification from an aE~I,ropliate biological sample (described, for example, in Shimonaka et al., Biochem.
~iophys. Res. Comm. 165:189-195, 1989) and expression in a suitable host, such as yeast(described, for exarnple, in Bayne et al., ~ Biol. C~em. 265: 15648- 15652, 1990).
In this regard, the IGF and binding proteins employed may be the native proteins, or 10 may contain modifications, such as the addition of label or the addition or deletion of sequences that have minim~l effect on the binding properties of the protein. Antibodies suitable for use in ELISA assays are commercially available from, for example? Amano Pharmaceutical Co. or may be raised against the IGF and/or binding protein of interest by any of a variety of techniques known to those of ordinary skill in the art. See, e.g., 15 Harlow and Lane, Antibodies. A l aboratory Man2~al, Cold Spring Harbor Laboratory, 1988. Monoclonal antibodies may be plepared, for example, using the technique ofKohler and Milstein, Eur. J. Immunol. 6:511 -519, 1976, and improvements thereto.
A secondary bioassay, based on the activity of the binding protein of interest, may also be employed for further characterization of a small molecule inhibitor 20 or other ligand inhibitor. For example, IGF-I stimulates 3T3 fibroblast proliferation in vi~ro. The addition of a binding protein inhibits this stimulation, and the level of inhibition can be deterrnined using [3H]thymidine incorporation assays well known to those of ordinary skill in the art. Molecules that are capable of reversing the binding protein inhibition are ligand inhibitors. Similar assays may be designed for use with 25 specific binding proteins based on the known biological properties of the binding proteins (e.g., the inhibition of granulosa cell steroidogenesis as described in Bicsak et al., Endocrinol. 126:2184-2189, 1990).
Animal models may be useful for further characterization of ligand inhibitors. For example, the effect of a ligand inhibitor on blood glucose levels may be 30 evaluated using ~nim~ such as rats. An inhibitor that normalizes blood glucose levels in hyperglycemic or diabetic animals may be useful for the treatment of diabetes.

CA 022248~9 1997-12-17 W O 97/39032 PCTrUS97/06503 Similarly, growth hormone deficient ~nim~l~ may be used to evaluate the utility of a ligand inhibitor for the treatment of conditions that respond to the ~fimini.~tration of ~ human growth horrnone, such as human growth hormone resistance.
Ligand inhibitors of the present invention may generally be used to 5 increase the level of free, biologically active IGF in a patient and to treat any of a variety of IGF-responsive conditions. The term "IGF-responsive condition"
encomr~ses any condition of a patient that may be alleviated or treated by the ~,lmini.stration of IGF, and includes diseases such as diabetes (insulin dependent, non-insulin dependent and type I/II), growth retardation, osteoporosis, human growth10 horrnone resistance, ALS, demyelinating diseases (including via remyelination), multiple sclerosis, muscular dystrophy, stroke, ophthalmic conditions, infertility, Alzheimer's disease and other dementias. The terrn "IGF-responsive condition" also encomp~cses states in which it is desirable to induce wound healing or bone repair, such as bone remodeling. As used herein, a "patient" refers to any warm-blooded 15 animal, preferably a human. A patient may or may not be afflicted with an IGF-responsive condition. Patients that are so afflicted may generally be identified through clinical diagnosis according to methods that are well known to those of ordinary skill in the art.
Within the context of the present invention, the minimum acceptable 20 increase in the level of free IGF is 10%, a preferred level is at least 50% and a particularly preferred level is at least ~0%. The level of free IGF may generally be determined by methods known in the art, such as resolving the plasma sample to different molecular size fractions on a Sephadex G-50 fine column developed in 0.02M
potassium phosphate buffer, pH 7.2. The free IGF-I with a molecular weight of 7.6 25 kDa will elute later than the larger molecular weight IGF-IIIGFBP complex. The free IGF-I fraction can then be quantitated by radioimmunoassay. Alternatively, bloodglucose levels may be used as an indirect measurement of free IGF levels.
Other techniques, such as MRI, PETSCAN, spect~c~nning or other similar im~ging techniques, may also be employed to measure the increase in the level 30 of free IGF. Some of these techniques use radiolabeled ligand to IGF binding proteins or IGF receptors. A preferred method is image analysis using PET positron-emitting CA 022248~9 1997-12-17 wO 97/39032 PCT/US97/06503 ligands (e.g., "C or '8F) of a single photon-emitting ligand (e.g, l23I-labeled ligand to IGF-binding proteins or IGF receptors). Free IGF levels are correlated to the amount of binding of the radiolabeled ligand. An increase in IGF levels is manifested by adecreased binding of the radiolabeled ligand to the IGF-binding proteins and IGF5 receptors. Within this im~ging technique, an increase in free IGF levels of about 10-30% or more is sufficient.
For ~-lmini~ration to a patient, the ligand inhibitors are preferably incorporated into pharmaceutical compositions, which comprise a therapeutically effective amount of one or more ligand inhibitors and a physiologically acceptable 10 carrier. A "physiologically acceptable carrier" may be any composition, carrier or diluent that is capable of aAmini~tration to a m~mm~l without producing undesirable physiological effects, such as nausea, dizziness or gastric upset. While any suitable carrier known to those of ordinary skill in the art may be employed in the pharmaceutical compositions of this invention, the type of carrier will vary depending 15 on the mode of allministration. In general, the pharmaceutical compositions may be ~lmini.~tered by injection (e.g., intracutaneous, intramuscular, intravenous or subcutaneous), intranasally (e.g, by aspiration) or orally. For parenteral a~lmini.~tration, such as subcutaneous injection, the carrier preferably comprises water, saline, alcohol, a fat, a wax and/or a buffer. For oral ~lmini.ctration, any of the above carriers and/or a 20 solid carrier, such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, sucrose, and magnesium carbonate, may be employed.
Small molecule inhibitors of the present invention are preferably capable of oral atlmini.stration in, for example, capsule or tablet form. Such ligand inhibitors have the advantage of decreased cost and increased convenience, as compared to 25 conventional treatments that rely on repeated intravenous injection. In general, the ability of a small molecule inhibitor to be a~mini~tered orally may be determined by in vivo assays. Such assays typically measure the decrease in the level of IGF bound to binding proteins in response to oral ~flmini~tration of the small molecule inhibitor. For example, the amount of small molecule inhibitor in the blood may be measured based 30 on its ability to inhibit binding of '25I-hlGF-I to IGF binding proteins. Blood samples may be drawn at various times after a~lmini.~tration of small molecule inhibitor or CA 022248~9 1997-12-17 W O 97139032 PCT~US97/06503 vehicle to ~nim~l~ or humans. If necessary, the ligand inhibitor may be extracted from the blood by standard procedures (e.g., ~0% acetonitrile/0.1% trifluoroacetic acid).
Briefly, 1 mL of the extraction solvent may be added to 200 IlL of plasma and the mixture is vortexed. The resultant precipitate may then be centrifuged at 12,000 x g for 5 5 minutes. The supernatant cont~ining the ligand inhibitor may then be Iyophilized and reconstituted in assay buffer (PBS cont~ining 0.2% Nonidet p 40TM) to yield a reconstituted extract. Blood samples or reconstituted extracts may then be used in a binding assay, such as that described in Example l, below. Ligand inhibitors capable of oral ~lm;ni~tration will show inhibition of binding of '25I-hIGF-I to IGF binding 10 proteins, relative to the vehicle control.
Routes and fre4uency of ~(lministration, as well as dosage, will vary depending on the ligand inhibitor and the desired in vivo response. A suitable dose is an amount of ligand inhibitor that, when a~1mini~tered as described above, is capable of improving the clinical outcome for a patient (e.g, fewer hypoglycemic episodes for 15 diabetic patients) in treated patients as compared to untreated patients.. ln general, for pharmaceutical compositions comprising one or more ligand inhibitors, the amount of each ligand inhibitor present in a dose ranges from about 0.1 to about 10, preferably from about 1 to about 3 mg/kg. A larger or smaller amount may, however, be employed, depending on the size of the ligand inhibitor. Treatments are typically 20 conducted one-two times per day, and may need to be continuous for retention of benefit. Patients may be monitored by assessing IGF levels as described above and by evaluating clinical symptoms.
A significant advantage of the present invention lies in the ability to vary the potency of the ph~rm~r.eutical composition and to target IGF effects to specific 25 tissues, minimi7ing side effects. The potency may be varied through the use of ligand inhibitors with varying abilities to inhibit the binding of IGF to one or more binding proteins. In this regard, the relative abilities of ligand inhibitors to inhibit binding may be evaluated by, for example, comparing the amount of inhibitor needed for half maximal displacement of specific binding in all in vitro binding assay as described 30 herein.

CA 022248~9 l997-l2-l7 W O 97139032 PCTfUS97/06503 IGF effects may be targeted by using ligand inhibitors that are specific for one or more particular binding proteins, and exploiting the relative tissue specificity of each of the six known binding proteins (see Rech}er, Vi~am~ns and Hormones 47:1-114, 1993). Binding protein-specific ligand inhibitors may be developed, as noted 5 above, by using different binding proteins within the binding assay described herein for the identification of small molecule inhibitors. Administration of such ligand inhibitors results in the release of IGF in only those tissues that contain the targeted binding proteins. For example, IGFBP-2, -4 and -6 are more prevalent in the brain. In addition, IGFBP-1 is present in the brain, although at lower concentrations. Small molecule 10 inhibitors that are specific for these binding proteins and are capable of crossing the blood/brain barrier (as discussed below) will release IGF in the brain, while leaving most of the serum IGF in its inactive form. Such inhibitors may be particularly useful for the treatment of ALS and other neurodegenerative diseases such as multiple sclerosis, demyelin~ting disease and Alzheimer's disease. Alternatively, peripheral 15 effects may be manipulated using primarily IGFBP-I, -3, -4, and -5. As noted above, circulating IGF-I may be released by inhibiting binding to IGFBP-3. In this regard, small molecule inhibitors that are not capable of crossing the blood/brain barrier are particularly well suited for providing peripheral effects.
The amount of a small molecule inhibitor that crosses the bloodlbrain 20 barrier may be assessed by techniques known to those of ordinary skill in the art, such as MRI, PETSCAN, spect~c~nnin~ or other similar im~gin~ techniques, some of which use radiolabeled ligand to IGF binding proteins or IGF receptors. As noted above, a preferred method is image analysis using PET positron-emitting ligands (e.g., "C or l8F) of a single photon-emitting ligand (e.g., '23I-labeled ligand to IGF-binding proteins 25 or IGF receptors). Decreased binding of the radiolabeled ligand to the IGF-binding proteins and IGF receptors indicates an increase in IGF levels. Such decreased binding is indicative of the level of small molecule inhibitor that has crossed the blood/brain barrier. Alternatively, IGF-I levels in the cerebrospinal fluid (CSF) can be measured by radioimmunoassay using commercially available assay kits (e.g, Peninsula 30 Laboratories, Inc., Belmont, CA) or by polyethylene/glycol precipitation, as described CA 022248~9 l997-l2-l7 in Example 1. An increase in the level of IGF-I in the CSF is indicative of an increase in the level of IGF-I in the brain.
In ~nim~l~, the blood/brain penetration of the small molecule inhibitors can be tested by ex vivo binding. Briefly, ~nim~ls may be injected (intravenously or 5 orally) with 15-50 mg/kg of a small molecule ligand inhibitor. The ~nim~l~ are then sacrif1ced at 15, 30, 60 and 90 minutes after ~-lmini.~tration of the drug. The brains are removed and homogenized in solubilization buffer (PBS cont~ining 0.2% Nonidet p 40TM detergent). The assay is then carried out by polyethyleneglycol pleci,oil~tion of the bound '2sI-hIGF/hIGFBP-3 complex. If any dru-g is present in the brain, it will 10 inhibit binding of the '25I-hIGF-I to the IGF binding proteins? resulting in fewer precipitated counts in the drug-treated animals than in :~nimz~ treated with vehicle alone.
Those of ordinary skill in the art will recognize that other tissues, and thus other IGF responsive conditions, may be targeted by ~-lmini~tering ligand 15 inhibitors specific for other binding proteins, or combinations thereof. The tissue distribution of the six known IGF binding proteins is presented in Table I, below:

Table I
Primary Tissue Distribution of IGF Bindin~ Proteins IGF Binding Protein Tissues BP- I placenta, liver BP-2 CSF, CNS, liver BP-3 ovary, adrenal, heart, kidney, liver, stomach, intestine BP-4 liver, brain cortex BP-5 liver, brain, lung, heart, spleen, stomach, kidney, adrenal, intestine BP-6 CSF and all tissue The following Examples are offered by way of illustration and not by way of limitation.

CA 022248~9 1997-12-17 W O 97139032 PCT~US97/06503 EXAMPLES

Example 1 5 Inhibition of IGF/BP-3 Bindin~ Usin~ hIGF-I Analo~s This Example illustrates the in vitro and in vivo inhibition of binding of IGF-I to BP-3, resulting in increased levels of free, biologically active IGF-I.
10 A. In vitro Bindin~ Assay The binding assay was performed in duplicate at room temperature in 0.2% BSA-PBS, pH 7.2. BP-3 was purified from rat serum as described in Shimonakaet al., ~iochem. Biophys. Res. Comm. 165:189-195, 1989. Two hundred microliters of a 2.5 nM BP-3 solution (0.5 pmol) was added to a 12 x 75-mm glass tube. The reaction 15 was started by the addition of an increasing concentration of unlabeled [T59]hIGF-I or [L24,S9,60,A31]hIGF-I (both prepared by chemical synthesis according to the procedure described in Shim~ki et al., J. Biol. Chem. 266:10646-10653, 1991) in 100 ~L followed by 100 ~L of '25I-labeled [T59]hIGF-I (30,000 cpm/~0.3 ng). After incubation for 2.5 hours, 100 ~L of 20% BSA and 500 IlL of 20% PEG-8000 in 20 phosphate buffered saline (PBS) was added and the mixture was vortexed and then centrifuged for 30 minutes at 3500 rpm. The supernatant was carefully removed bysuction and the pellet counted in a gamma counter.
As shown in Figure 1, 0.5 pmol of IGFBP-3 could bind 23.3% of the '2sI-labeled IGF-I tracer, and the non-specific binding was 1.8%. Half-maximal 25 displacement of the specific binding was achieved at approximately 0.32 pmol [T59~hIGF-I per tube and 0.8 pmol ~L24,59,60,A31]hlGF-I per tube (Figure 1).
This assay may also be employed to screen for small molecule ligand inhibitors.

CA 022248~9 1997-12-17 W O 97/39032 PCTrUS97/06S03 B. r3Hl-ThYmidine Incorporation Assay BALB/c 3T3 cells were trypsanized and diluted to 50,000 cells per mL
in 10% calf serum-DMEM. The cells were aliquoted to 96-well microtiter plates (180 ~L/well). After 48 hours incubation at 37~C and 5% CO2, the plates were washed twice S with 0.1% calf serum-DMEM and incubated for an additional 24 hours. To each well, 20 ~L of sample(s) cont~ining IGF analog and/or binding protein and I ~LCi [3H]-thymidine were added and the plates were incubated for precisely 24 hours. After the incubation, the medium was removed and the cells were fixed by adding 200 IlL of25% acetic acid-75% ethanol per well. After removal of the fixing solution the plates 10 were washed three times with cold 10% TCA and the cells in each well were lysed in 200 ~L 0.2M NaOH. The entire 200 IlL of lysed solution was transferred into a scintillation vial and 2.5 mL scintillation liquid was added and the vials counted in a ,~-counter.
[T59]hIGF-I dose dependently stimulated proliferation, as measured by 15 DNA synthesis, in 3T3 cells with a ED~o of 10-20 nM (Figure 2), whereas [L24,59,60,A31]hIGF-I did not induce any DNA synthesis in 3T3 cells with a dose as high as 8,000 nM (Figure 2). After incubation with 10 nM [T59]hIGF-I, the incorporated [3H]-thymidine in the 3T3 cells was increased 4-fold in comparison with the control (Figure 3). Incubation with 25 nM IGFBP-3 completely inhibited the [3H]-20 thymidine incorporation induced by 10 nM [T59]hIGF-I ~Figure 3~. Addition of [L24,59,60,A31]hIGF-I (which only binds to IGFBP-3, and not to the IGF-I receptors) could totally reverse the blocking effect of 25 nM IGFBP-3 to release the [T59~hIGF-I
bioactivity with a EDso of about 25 nM [L24,59,60,A31]hIGF-I (Figure 3).
This assay may also be employed to evaluate the biological activity of 25 small molecule ligand inhibitors.

CA 022248~9 1997-12-17 W O 97/39032 PCT~US97/06503 Example 2 Norm~1i7~tion of Blood Glucose Levels in Hyper~lvcemic Rats usin~ IGFBP-3 Inhibitor This Exarnple illustrates the use of ligand inhibitors of IGFBP-3 for the treatment of ~nim~ with elevated blood glucose.
Rats were made hyperglycemic with an intraperitoneal injection of glucose. The ~L24,59,60,A31]hIGF-I analog (noted as IGFBP3-LI) (S~lg/min) was infused intravenously for 40 minutes and blood glucose levels were monitored before, 10 during and after the infusion. As a control, bovine insulin was infused at 1 ~Lg/min for 40 minutes and blood glucose was monitored at the sarne time points.
As depicted in Figure 5, while the insulin dramatically lowered blood glucose levels below the normal baseline, making the Anim~ hypoglycemic, the [L24,59,60,A31]hIGF-I analog surprisingly normalized blood glucose levels. In a 15 related experiment, insulin dramatically decreased blood glucose levels on a normal fasted blood glucose baseline, but [L24,59,60,A31]hIGF-I had no effect.
These results indicate that [L24,59,60,A31]hIGF-I can normalize blood glucose levels in animals with elevated blood glucose but, unlike insulin, this analog does not decrease the blood glucose levels below the normal baseline and make the 20 ~nimz~ hypoglycemic. Thus, the use of inhibitors of IGF binding to IGFBP-3 have distinct advantages over insulin for the treatment of diabetes. Diabetics receiving insulin normally experience a rebound effect after intravenous insulin injection such that blood sugar levels fall below normal levels. The inhibitors of the present invention normalize blood sugar levels without the risk of hypoglycemia. In addition, both25 insulin dependent and non-insulin dependent diabetics may be treated.

CA 022248~9 1997-12-17 W 097/39032 PCTfUS97106S03 ExamPle 3 Norm~li7~tion of Blood Glucose Levels in Diabetic Mice using IGFBP-3 Inhibitor S This Example illustrates the use of ligand inhibitors of IGFBP-3 for the treatment of diabetes in ~nim~
Two severely diabetic female NOD mice with blood glucose levels of 450-500 mg/dl were treated with the [L24,59,60,A31]hIGF-I analog. These mice were severely diabetic and were expected to die within two days without treatment. The 10 [L24,59,60,A31]hlGF-I analog was a~mini.~tered at time 0 (25 ~Lg/animal). at 30 minutes (50 ~Lg/animal) and at 60 min (100 ~lg/animal) by tail vein injection inphysiological saline). Blood glucose levels were monitored using a glucometer before the initial injection and throughout the time course of the experiment.
In both ~nim~l~, the blood glucose levels decreased dramatically to 250 15 mg/dl after 80 minutes (Figure 4). Since the threshold for normal blood glucose is 220 mg/dl, the blood glucose levels were almost normalized in both ~nim711~. These results demonstrate that the [L24,59,60,A31]hIGF-I analog may be used to lower blood glucose levels in the treatment of diabetes.

From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein for the purpose of illustration, various modifications may be made without deviating from the spirit and scope of the invention.

CA 022248~9 1997-12-17 W O 97/39032 PCTrUS97106503 SEQUENCE LISTING

(1) GENERAL INFORMATION:
(i) APPLICANTS: DOMINIC P. BEHAN, NICHOLAS LING, XIN-JUN LIU AND
AMITABH GAUR
(ii) TITLE OF INVENTION: LIGAND INHIBITORS OF INSULIN-LIKE GROWTH
FACTOR BINDING PROTEINS AND METHODS OF USE THEREFOR
(iii) NUMBER OF SEQUENCES: 2 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: SEED and BERRY LLP
(B) STREET: 6300 Colu~bia Center. 701 Fifth Avenue (C) CITY: Seattle (D) STATE: Washington (E) COUNTRY: USA
(F) ZIP: 98104-7092 (v) COMPUTER READABLE FORM.
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (vi) CURRENT APPLICATION DATA:
(A) APPLICATTON NUMBER:
(B) FILING DATE: 17-APR-1996 (C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Maki, David J.
(B) REGISTRATION NUMBER: 31,392 (C) REFERENCE/DOCKET NUMBER: 690068.425 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (206) 622-4900 (B) TELEFAX: (206) 682-6031 (2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:

CA 022248~9 1997-12-17 W 097/39032 PCT~US97/06503 (A) LENGTH: 70 amino acids (B) TYPE: amino acid (C~ STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
Gly Pro Glu Thr Leu Cys Gly Ala Glu Leu Val Asp Ala Leu Gln Phe Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Tyr Gly Ser Ser Ser Arg Arg Ala Pro Gln Thr Gly Ile Val Asp Glu Cys Cys Phe Arg Ser Cys Asp Leu Arg Arg Leu Glu Met Tyr Cys Ala Pro Leu Lys Pro Ala Lys Ser Ala (2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 70 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Gly Pro Glu Thr Leu Cys Gly Ala Glu Leu Val Asp Ala Leu Gln Phe Val Cys Gly Asp Arg Gly Phe Leu Phe Asn Lys Pro Thr Gly Ala Gly Ser Ser Ser Arg Arg Ala Pro Gln Thr Gly Ile Val Asp Glu Cys Cys Phe Arg Ser Cys Asp Leu Arg Arg Leu Glu Leu Leu Cys Ala Pro Leu Lys Pro Ala Lys Ser Ala

Claims

1. A ligand inhibitor that inhibits the binding of an insulin-like growth factor to one or more insulin-like growth factor binding proteins, for use within a method for increasing the level of free, biologically active insulin-like growth factor in a patient.

2. The ligand inhibitor of claim 1 wherein the ligand inhibitor is [L24,59,60,A31]hIGF-I or a variant thereof that differs only in conservative substitutions and/or modifications.

3. The ligand inhibitor of claim 1 wherein the ligand inhibitor is a small molecule inhibitor.

4. The ligand inhibitor of claim 1 wherein the level of free, biologically active insulin-like growth factor increases in the patient's blood.

5. The ligand inhibitor of claim 1 wherein the level of free, biologically active insulin-like growth factor increases in the patient's brain.

6. The ligand inhibitor of claim 1 wherein the insulin-like growth factor is IGF-I.

7. The ligand inhibitor of claim 1 wherein the insulin-like growth factor is IGF-II.

8. The ligand inhibitor of claim 1 wherein the insulin-like growth factor binding protein is IGFBP-3.

9. The ligand inhibitor of claim 1 wherein the insulin-like growth factor binding protein is selected from the group consisting of IGFBP-1, IGFBP-2, IGFBP-4, IGFBP-5, IGFBP-6 and combinations thereof.

10. A ligand inhibitor that inhibits the binding of an insulin-like growth factor to one or more insulin-like growth factor binding proteins, for use within a method for treating an IGF-responsive condition in a patient.

11 . The ligand inhibitor of claim 10 wherein the ligand inhibitor is [L24,59,60,A31]hIGF-I or a variant thereof that differs only in conservative substitutions and/or modifications.

12. The ligand inhibitor of claim 10 wherein the ligand inhibitor is a small molecule inhibitor.

13. The ligand inhibitor of claim 10 wherein the insulin-like growth factor is IGF-I.

14. The ligand inhibitor of claim 10 wherein the insulin-like growth factor is IGF-II.

15. The ligand inhibitor of claim 10 wherein the insulin-like growth factor binding protein is IGFBP-3.

16. The ligand inhibitor of claim 10 wherein the insulin-like growth factor binding protein is selected from the group consisting of IGFBP-1, IGFBP-2, IGFBP-4, IGFBP-5, IGFBP-6 and combinations thereof.

17. The ligand inhibitor of claim 10 wherein the IGF-responsive condition is selected from the group consisting of diabetes, growth retardation osteoporosis, human growth hormone resistance, wounds, bone damage, ALS, Alzheimer's disease, demyelinating disease, multiple sclerosis, muscular dystrophy, stroke and neuronal degeneration.

18. A pharmaceutical composition, comprising:
(a) one or more ligand inhibitors that inhibit the binding of an insulin-like growth factor to one or more insulin-like growth factor binding proteins; and (b) a physiologically acceptable carrier.

19. A pharmaceutical composition according to claim 18 wherein the ligand inhibitor is [L24,59,60,A31]hIGF-I or a variant thereof that differs only in conservative substitutions and/or modifications.

20. A pharmaceutical composition according to claim 18 wherein the ligand inhibitor is a small molecule inhibitor.

21. A method for screening for a small molecule inhibitor that inhibits binding of an insulin-like growth factor to an insulin-like growth factor binding protein, comprising:
(a) combining an insulin-like growth factor with an insulin-like growth factor binding protein in a solution containing a candidate small molecule, such that the binding protein and the growth factor are capable of forming a complex; and (b) determining the amount of complex in the solution, relative to a predetermined level of binding in the absence of the small molecule, and therefrom evaluating the ability of the small molecule to inhibit binding of an insulin-like growth factor to an insulin-like growth factor binding protein.

22. The method of claim 21 wherein the insulin-like growth factor is IGF-I.

23 . The method of claim 21 wherein the insulin-like growth factor binding protein is IGFBP-3.

24. The method of claim 21 wherein the insulin-like growth factor binding protein is selected from the group consisting of IGFBP-1, IGFBP-2, IGFBP-4, IGFBP-5, IGFBP-6 and combinations thereof.

25. A ligand inhibitor that inhibits the binding of the protein to one or more insulin-like growth factor binding proteins, for use within a method for increasing the level of a free, biologically active protein in a patient.

26. A ligand inhibitor that inhibits the binding of insulin-like growth factor to one or more insulin-like growth factor binding proteins, for use within a method for increasing the level of free IGF-II in a patient.