EP1117425A2

EP1117425A2 - Methods for the treatment of non-thyroid disorders

Info

Publication number: EP1117425A2
Application number: EP99951681A
Authority: EP
Inventors: Desmond Mascarenhas
Original assignee: Celtrix Pharmaceuticals Inc
Current assignee: Celtrix Pharmaceuticals Inc
Priority date: 1998-10-02
Filing date: 1999-09-29
Publication date: 2001-07-25
Also published as: WO2000020024A2; WO2000020024A3; CA2345642A1; AU6407099A; JP2002526417A

Abstract

Methods are disclosed for the treatment of non-thyroid disorders which respond to IGF, respond to IGFBP-3, or which are IGF-dependent. Thyroid axis agonists and IGF are administered to subjects suffering from non-thyroid disorders which respond to IGF, alleviating the symptoms of the disorders. Thyroid axis antagonists and IGFBP-3 are administered to subjects suffering from non-thyroid disorders which respond to IGFBP-3, alleviating the symptoms of the disorders. Thyroid axis antagonists are administered to subjects suffering from IGF-dependent non-thyroid disorders, thereby alleviating the symptoms of the disorders.

Description

METHODS FOR THE TREATMENT OF NON-THYROID DISORDERS

TECHNICAL FIELD The invention relates generally to the field of medical treatment, and more particularly to methods for modulating the actions of insulin-like growth factors (IGFs) and/or insulin-like growth factor binding proteins (IGFBPs).

BACKGROUND

The thyroid hormones, triiodothyronine (T3) and tetraiodothyronine (T4) are major metabolic regulators in mammals. T4 is less active than T3, and can be converted to T3 in peripheral tissues. Administration of T4 or T3 increases metabolism, erythropoiesis, bone turnover and the rate of muscle relaxation. Although thyroid hormones increase the rate of protein synthesis, hyperthyroidism is associated with weight loss and muscle wasting. Hypothyroidism can be accompanied by lethargia, decreased pulmonary function (hypo ventilation), low cardiac output, and decreased renal output. The thyroid hormones also interact with other endocrine hormones, including the growth hormone axis and steroid hormones.

T4 and T3 are synthesized from thyroglobulin, a protein that is iodinated on its tyrosine residues. Two iodinated tyrosines are condensed to form a molecule of T4 or T3. Thyroglobulin, which is stored extracellularly in the follicular lumen of the thyroid gland, acts as a storage molecule for the iodinated tyrosine residues. Iodinated tyrosine residues are released from thyroglobulin by intracellular proteolysis in thyroid cells. IGF -I has been shown to increase transcription of thyroglobulin in FRTL-5 (rat thyroid) cells (Kamikubo et al. (1990) Mol. Endocrinol, 4:2021-2029). The influence of increased levels of thyroglobulin mRNA on T4 and T3 levels is, however, unknown. IGF-I and IGF-II are growth factors that have related amino acid sequence and structure, with each polypeptide having a molecular weight of approximately 7.5 kilodaltons (Kd). IGF-I mediates the major effects of growth hormone, and thus is the primary mediator of growth after birth. IGF-I has also been implicated in the actions of various other growth factors, since treatment of cells with such growth factors leads to increased production of IGF-I. In contrast, IGF-II is believed to have a major role in fetal growth. Both IGF-I and IGF-II have insulin-like activities (hence their names), and are mitogenic (stimulate cell division) and/or are trophic (promote recovery/survival) for cells in neural, muscular, reproductive, skeletal and other tissues. IGF-I has also been shown to induce DNA synthesis in FRTL-5 cells, a diploid nontransformed line of rat thyroid follicular cells, an activity which is potentiated by thyroid stimulating hormone (TSH)

(Yamamoto et al., 1996, Endocrinology 137(5):2036-2042). Additionally, some tumors have also been shown to be insulin-dependent for their continued growth.

Unlike most growth factors, IGFs are present in substantial quantity in the circulation, but only a very small fraction of this IGF is free in the circulation or in other body fluids. Most circulating IGF is bound to the IGF -binding protein IGFBP-3. IGF-I may be measured in blood serum to diagnose abnormal growth-related conditions, e.g., pituitary gigantism, acromegaly, dwarfism, various growth hormone deficiencies, and the like. Although IGF-I is produced in many tissues, most circulating IGF-I is believed to be synthesized in the liver. IGF is known to bind to at least three different cellular receptors; the type 1 IGF receptor, the type 2 IGF receptor, and the insulin receptor (to which IGF binds with much lower affinity than the type 1 or 2 receptor). Mutants of IGF-I have been described which have altered binding to one or more of these cellular receptors. Mutations at residue 24 (normally tyrosine) to non-aromatic residues or replacement of residues 28-37 selectively affects binding to the type 1 receptor, while mutations at residues 49-51 can selective reduce type 2 receptor binding. Mutations at residue 60 (from tyrosine to non-aromatic amino acids) can alter binding to the type 1 and 2 IGF receptors as well as the insulin receptor (Cascieri et al. (1988) Biochemistry 27:3229-3233; Cascieri et al. (1989) JBiol. Chem. 264:2199-2202; Bayne et al. (1988) JBiol. Chem. 264:11004-11008; Bayne et al. (1990) J. Biol. Chem. 265:15648-15652).

Almost all IGF circulates in a non-covalently associated ternary complex composed of IGF-I or IGF-II, IGFBP-3, and a larger protein subunit termed the acid labile subunit (ALS). The IGF/IGFBP-3/ALS ternary complex is composed of equimolar amounts of each of the three components. ALS has no direct IGF binding activity and appears to bind only to the IGF/IGFBP-3 binary complex. The IGF/IGFBP-3/ALS ternary complex has a molecular weight of approximately 150 Kd. This ternary complex is thought to function in the circulation "as a reservoir and a buffer for IGF-I and IGF-II preventing rapid changes in the concentration of free IGF" (Blum et al., pp. 381-393, MODERN CONCEPTS IN INSULIN-LIKE GROWTH FACTORS (E.M. Spencer, ed., Elsevier, New York, 1991).

Some mutant IGF-Is exhibit altered binding to IGFBP-3 and/or alterations in the ability to form the ternary complex. For example, mutations at residues 3, 4, 8, 9, 12, 15,

16 and 24 (B domain mutants) and mutations at residues 49-51 reduce formation of the binary complex, while mutations involving residues 55 and 56, as well as IGF-I where residues 63-70 (1-62 IGF-I) were deleted or residues 28-37 were replaced with a Gly₄ linker (l-27-Gly₄-38-70 IGF-I) or where both changes were made (l-27-Gly₄-38-62 IGF-I)) and mutants thereof (for example, 1-62 IGF-I where residue 24 was also changed) actually have increased binding to IGFBP-3. Some of these mutants, particularly the 1-62 IGF-I with a mutation at residue 24, have a reduced capacity for formation of the ternary complex, even though formation of the binary complex is increased (Baxter et al. (1992) J Biol. Chem. 267:60-65). Nearly all of the IGF-I, IGF-II and IGFBP-3 in the circulation is in complexed form, so very little free IGF is detected. Moreover, a high level of free IGF in blood is undesirable. High blood levels of free IGF would lead to serious hypoglycemia due to the insulin-like activities of IGF. In contrast to the IGFs and IGFBP-3, there is a substantial pool of free ALS in plasma which assures that IGF/IGFBP-3 complex entering the circulation immediately forms the ternary complex.

IGFBP-3 is the most abundant IGF binding protein in the circulation, but at least five other distinct IGF binding proteins (IGFBPs) have been identified in various tissues and body fluids. Although these proteins bind IGFs, they each originate from separate genes and have unique amino acid sequences. Thus, the binding proteins are not merely analogs or derivatives of a common precursor. Unlike IGFBP-3, the other IGFBPs in the circulation are not saturated with IGFs. Moreover, none of the IGFBPs other than IGFBP-3 can form the 150 Kd ternary complex.

IGF-I and IGFBP-3 may be purified from natural sources or produced by recombinant means. For instance, purification of IGF-I from human serum is well known in the art (Rinderknecht et al. (1976) Proc. Natl. Acad. Sci. USA 73:2365-2369).

Production of IGF-I by recombinant processes is shown in EP 0 128 733, published in December of 1984. IGFBP-3 may be purified from natural sources using a process such as that shown in Baxter et al. (1986, Biochem. Biophys. Res. Comm. 139:1256-1261). Alternatively, IGFBP-3 may be synthesized by recombinantly as discussed in Sommer et al, pp. 715-728, MODERN CONCEPTS OF INSULIN-LIKE GROWTH FACTORS (E.M. Spencer, ed., Elsevier, New York, 1991). Recombinant IGFBP-3 binds IGF -I in a 1 :1 molar ratio.

Topical administration of IGF-I/IGFBP-3 complex to rat and pig wounds is significantly more effective than administration of IGF-I alone (Id.). Subcutaneous administration of IGF-I/IGFBP-3 complex to hypophysectomized, ovariectomized, and normal rats, as well as intravenous administration to cynomolgus monkeys, "substantially prevents the hypoglycemic effects" of IGF-I administered alone (Id.).

IGF has been proposed as a treatment for a wide variety of indications. U.S. Patents Nos. 5,434,134, 5,128,320, 4,988,675, 5,106,832, 5,534,493, 5,202,119 and 5,273,961 and have disclosed the use of IGF for the treatment of cardiomyopathy and myocardial infarction, steroid-induced catabolism, type II (insulin resistant) diabetes, renal disorders, pancreatic disorders, for increasing humoral immune response and for prevention of acute renal failure, respectively. Additionally, European Patents Nos. EP 434 625, EP 436469 and EP 560 723 and International Patent Applications Nos. WO 93/23071, WO 91/12018, WO 92/00754, WO 93/02695 and WO 93/08826 disclose the use of IGF for the treatment of bone disorders, type I (juvenile or insulin-responsive) diabetes and gastrointestinal disorders.

The use of IGF complexed with IGFBP-3 has also been described for use in the treatment of a variety of conditions. U.S. Patents Nos. 5,200,509, 5,681,818, 5,187,151, 5,407,913, and 5,527,776 disclose the use of IGF/IGFBP-3 complex for the treatment of osteoporosis, for inducing an anabolic state when given by subcutaneous bolus injection, for increasing tissue repair when given systemically, and for treating anemia. International

Patent Applications Nos. WO 95/03817, WO 95/08567, WO 95/13823, WO 95/13824, WO 96/02565 disclose the use of IGF/IGFBP-3 complex for the treatment of disorders of the reproductive, immunologic, neural, renal, and skeletal systems.

In addition to its activities in other organ systems, IGF has trophic effects on the cells of the peripheral and central nervous system. IGF's trophic effects on neural cells include promoting the survival of a variety of neuronal cell types as well as promoting neurite outgrowth in motor neurons. U.S. Patents Nos. 5,093,317, 5,420,112, 5,068,224, and International Patent Applications Nos. WO 93/02695, WO 93/08826 and WO 95/13823 describe the use of IGF or IGF complexed to IGFBP-3 for the treatment of disorders of the nervous system, exploiting IGF's trophic activity on the cells of nervous tissues.

Other disorders exist which would benefit from a reduction in the levels of IGF. For example, some cancer cells are dependent on IGF for continued survival (Resnicoff et al. (1994) Cancer Res. 54:2218-2222). Reduction in circulating IGF levels could result in tumor progression, as IGF-dependent tumor cells undergo apoptosis. In autoimmune disorders, reduction in IGF levels could reduce symptoms of these disorders, as IGF is known to have stimulatory effects on the immune system.

In addition to its role as the major carrier protein for IGF in serum, IGFBP-3 has been recently shown to have a number of different activities. IGFBP-3 can bind to an as- yet unidentified molecule on the cell surface, where it can inhibit the activity of exogenously-added IGF-I (Karas et al, 1997, J. Biol. Chem. 272(26):16514-16520).

Although the binding of IGFBP-3 to cell surfaces can be inhibited by heparin, the unidentified cell surface binding molecule is unlikely to be a heparin-like cell surface glycosaminoglycan, because enzymatic removal of heparin glycosaminoglycans has no effect on IGFBP-3 cell surface binding (Yang et al., 1996, Endocrinology 137(10):4363- 4371). It is not clear if the cell surface binding molecule is the same or different than the

IGFBP-3 receptor that was identified by Leal et al. (1997, J. Biol. Chem. 272(33):20572- 20576), which is identical to the type V transforming growth factor-beta (TGF-β) receptor.

IGFBP-3 has also been found to promote apoptosis. Interestingly, IGFBP-3 has been shown to promote apoptosis in cells with and without functional type 1 IGF receptors (Nickerson et al., 1997, Biochem. Biophys. Res. Comm. 237(3):690-693; Rajah et al, 1997,

J. Biol. Chem. 272(18):12181-12188). However, there are conflicting reports as to whether apoptosis is induced by full length IGFBP-3 or a proteolytic fragment of IGFBP-3 (Rajah et al., ibid; Zadeh et al., 1997, Endocrinology 138(7):3069-3072).

Like most protein-based therapeutics, IGF, IGFBP, and particularly IGF/IGFBP complex, are expensive to produce. Additionally, as with all drugs, high doses of IGF can lead to serious adverse effects. Accordingly, there is a need in the art for enhancing the actions of IGF, IGFBP, and IGF/IGFBP complex.

Because certain disorders are caused or associated with excess IGF, there is also a need in the art for compounds which can inhibit or reduce the activity of IGFs or which can enhance the action of IGF-inhibiting compounds.

DISCLOSURE OF THE INVENTION The invention provides methods for modulating the effects of insulin-like growth factors (IGFs) on non-thyroid cells by administering IGF and compounds which affect the thyroid hormone axis. IGF and IGFBP, alone or in combination, can stimulate proliferation of a variety of cell types, act as anti-apoptotic agents, or stimulate certain tissues, such as the immune and renal systems. Administration of compounds which modulate the thyroid hormone axis modulates the effects of exogenous and endogenous IGF and/or IGFBP. In one embodiment, the invention provides methods for treating non-thyroid disorders which respond to IGF by administering thyroid axis agonists and IGF or

IGF/IGFBP. A thyroid axis agonist is administered with IGF, reducing or alleviating symptoms of the non-thyroid disorder responsive to IGF. Alternately, the thyroid axis agonist is administered with IGF/IGFBP complex. Administration of a thyroid axis agonist along with IGF or IGF/IGFBP-3 complex enhances the actions of IGF, thereby alleviating the symptoms of the IGF-responsive non-thyroid disorder.

In another embodiment, the invention provides methods for treating non-thyroid disorders which respond to IGFBP-3. IGFBP-3 and a thyroid axis antagonist are administered to subjects having a disorder which responds to IGFBP-3. Administration of a thyroid axis antagonists with IGFBP-3 enhances the anti-mitotic and pro-apoptotic activities of IGFBP-3, thereby alleviating the symptoms of non-thyroid disorders which respond to IGFBP-3. In accordance with the methods of this embodiment, IGFBP-3 may be administered as a complex of IGFBP-3 and mutant IGF-I.

In a further embodiment, the invention provides methods for treating IGF- dependent, non-thyroid disorders. A thyroid axis antagonist is administered to a subject having an IGF-dependent, non-thyroid disorder. Administration of thyroid axis antagonists alleviates the symptoms of IGF-dependent, non-thyroid disorders. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the amino acid sequence of native human IGF-I. FIG. 2 shows the amino acid sequence of the naturally occurring Ala₅ allele of native human IGFBP-3.

BEST MODE FOR CARRYING OUT THE INVENTION Provided herein are improved methods for treating non-thyroid disorders responsive to IGF or IGFBP-3. Symptoms of disorders which respond to IGF are alleviated by administration of a thyroid axis agonist and IGF or IGF/IGFBP-3 complex. The symptoms of disorders associated with excess IGF are alleviated by administration of a thyroid axis antagonist. Additionally, the symptoms of disorders which respond to IGFBP are alleviated by the administration of IGFBP and a thyroid axis antagonist.

Definitions As used herein, "IGF" refers to insulin-like growth factor from any species. IGF includes both IGF-I and IGF-II, in native-sequence or variant form, including but not limited to naturally-occurring allelic variants. IGF may be from any source, whether natural, synthetic or recombinant.

As used herein, "IGF-I" refers to insulin-like growth factor I from any species, including bovine, ovine, porcine and human, in native-sequence or variant form, including but not limited to naturally-occurring allelic variants. IGF-I may be from any source, whether natural, synthetic or recombinant, provided that it will bind IGFBP-3 at the appropriate site and bind to and stimulate the type 1 IGF receptor. Preferred herein is human native-sequence, mature IGF-I, preferably without an amino-terminal methionine. More preferably, the native sequence, mature IGF-I is produced recombinantly, for example, as described in PCT publication WO 95/04076.

As used herein, the term "mutant IGF-I" refers to which have altered amino acid sequences at one or more sites in the molecule. Mutant IGF-I retains its ability to bind IGFBP-3, but may be altered in its other properties, such as binding to the type I or type II IGF receptor or binding to the insulin receptor. Descriptions of mutant IGF -Is may be found in Cascieri et al. (1988) Biochemistry 27:3229-3233; (1989) J. Biol. Chem. 264:2199-2202), Bayne et al. (1990) J. Biol. Chem. 265:15648-15652) and Baxter et al. (1992) J. Biol. Chem. 267:60-65). Examples of mutant IGF-I include mutants in which one or more of IGF -Is tyrosine residues (i.e., residues 24, 31, or 60) are replaced with non- aromatic residues (i.e., other than tyrosine, phenylalanine or tryptophan), mutants where amino acid residues 49, 50, 51, 53, 55 and 56 are altered (for example, where residues 49- 50 are altered to Thr-Ser-Ile or where residues 55-56 are altered to Tyr-Gln), and combinations thereof.

As used herein, "IGF-II" refers to insulin-like growth factor II from any species, including bovine, ovine, porcine and human, in native-sequence or variant form, including but not limited to naturally-occurring allelic variants. IGF-II may be from any source, whether natural, synthetic or recombinant, provided that it will bind IGFBP-3 at the appropriate site. Preferred herein is human native-sequence, mature IGF-II, preferably without an amino-terminal methionine. More preferably, the native sequence, mature IGF- I is produced recombinantly, for example, as described in PCT publication WO 95/04076.

As used herein, "IGFBP-3" refers to insulin-like growth factor binding protein 3. IGFBP-3 is a member of the insulin-like growth factor binding protein family. IGFBP-3 may be from any species, including bovine, ovine, porcine and human, in native-sequence or variant form, including but not limited to naturally-occurring allelic variants (e.g. , the Ala₅ and Gly₅ variants of native human IGFBP-3). Preferred IGFBP-3 embodiments include native sequence human IGFBP-3 and variants of human IGFBP-3 wherein the one or more of the asparagine residues which form the normal N-linked glycosylation sites (positions 89, 109 and 172) are changed to aspartate (e.g. : N89D; N109D; N172D; N89D,N109D; N89D,N172D; N109D,N172D; and N89D,N109D,N172D variants) or to other amino acid residues (e.g. : N89X; N109X; N172X; N89X,N109X; N89X,N172X;

N109X,N172X; and N89X,N109X,N172X variants) as well as variants which have been altered to improve resistance to degradation, such as alterations at positions 116 and 135 (e.g., Dl 16E, D135E and Dl 16E,D135E), or alterations which affect the nuclear localization signal (NLS) of IGFBP-3, which is located at residues 215 through 232 (Radulescu, 1994, Trends Biochem Sci. 19(7):278). Examples of preferred NLS variant

IGFBP-3s include K228E, R230G, K228E,R230G, K228X, R230X, and K228X,R230X, as well as variations at residues 215, 216 and 231. Of course, a variant IGFBP-3 may include more than one type of variation (e.g., a variant IGFBP-3 may be both ND variant and degradation resistant variant). IGFBP-3 can form a binary complex with IGF, and a ternary complex with IGF and the acid labile subunit (ALS). IGFBP-3 may be from any source, whether natural, synthetic or recombinant, provided that it will bind IGF-I and ALS at the appropriate sites. Preferably, IGFBP-3 is produced recombinantly, as described in PCT publication WO 95/04076.

The term "non-thyroid disorder which responds to IGF", as used herein, refers to any disorder which may be treated with IGF which is not a disorder of the thyroid. IGF has a variety of effects including, but not limited to, anabolic effects on the musculo-skeletal system, anti-apoptotic effects on neuronal cells, mitogenic and stimulatory effects on cells of the immune system, and stimulatory, trophic, and mitogenic effects on kidney cells. Accordingly, a non-thyroid disorder which responds to IGF is a disorder which benefits from the activities of IGF other than a disorder of the thyroid, such as: neurological disorders such as amyotrophic lateral sclerosis, Charcot-Marie-Tooth Syndrome, diabetic neuropathy, and drug-induced neuropathy (such as peripheral neuropathy induced by chemotherapeutic agents including vincristine, cisplatin, and the like), and pulmonary disorders such as chronic obstructive pulmonary disease; renal disorders such as glomerulonephritis, glomerulosclerosis, interstitial nephritis, acute tubular necrosis, diabetic nephropathy, autoimmune nephropathy, and acute and chronic renal failure; growth disorders such as growth hormone insufficiency, hypopituitarism, growth hormone resistance and Laron dwarfism; recovery from bodily insults, -such as recovery from trauma, burns, bone fractures or surgery; gastrointestinal disorders such as short bowel syndrome and pancreatic disease; reversal of catabolism in subjects with acquired immune deficiency syndrome (AIDS), cancer cachexia, or steroid-induced catabolism (such as can occur as a result of long term steroid therapy for disorders such as asthma, autoimmune disease, inflammatory bowel disease, immune suppression for organ transplantation, and rheumatoid diseases); bone disorders such as osteoporosis, osteopetrosis, osteogenesis imperfecta, and Paget's disease; reproductive disorders such as hypogonadotropic hypogonadism, male infertility, failure of gamete maturation, and polycystic ovarian disease; hematopoietic disorders such-as anemia, plasma cell dyscrasias, erythropoietin insensitivity, and deficient total hemoglobin; and disorders of glucose homeostasis, including type I diabetes mellitus (IDDM), type II diabetes mellitus (NIDDM), and insulin- resistant diabetes.

The term "non-thyroid disorder which responds to IGFBP-3", as used herein, refers to any disorder for which the symptoms are alleviated by treatment with IGFBP-3 which is not a disorder of the thyroid. IGFBP-3 has been shown to have a receptor separate from the type 1 and 2 IGF receptors and has been shown to bind to cell surfaces, where it can inhibit the action of exogenous IGF. Additionally, IGFBP-3 has been shown to induce apoptosis in several cancer cell lines. Accordingly, a disorder which responds to IGFBP-3 is any disorder which would benefit from the effects of IGFBP-3 administered in the absence of

IGF. Disorders which respond to IGFBP include cancer, including but not limited to prostate, breast, colon and lung carcinomas, acromegaly, diabetic retinopathy, keloid scars, malignant exophthalmos, and other disorders as are known in the art.

A "IGF-dependent, non-thyroid disorder" is a disorder which is associated with excess levels of IGF. IGF is a mitogen for many cell types and has trophic (particularly anti-apoptotic) activities as well. Accordingly, an IGF-dependent disorder which is not a thyroid disorder is a proliferative disorder in which IGF stimulates or otherwise peφetuates the disorder (by, for example, preventing apoptosis in cells which cause the disorder). Such conditions include cancer, including but not limited to prostate, breast, colon and lung carcinomas, proliferative diabetic retinopathy, and the like.

A "thyroid axis agonist" is a compound which acts to increase thyroid hormone activity in a subject. A thyroid axis agonist may be a thyroid gland stimulant or a thyroid receptor agonist. Thyroid receptor agonists may be in the form of free acids or bases, or may be pharmaceutically acceptable salts of the free acids or bases. Thyroid gland stimulants include indirect stimulators of the thyroid gland, such as thyrotropin releasing hormone (TRH), TRH analogs and iodinated compounds such as iodinated casein, or it may be a direct stimulator of the thyroid gland, including thyroid stimulating hormone (TSH) and analogs thereof. A thyroid receptor agonist is a compound which stimulates the thyroid receptor, such as 3,3'-5,5'-L-thyroxine (T4), 3,3'-5-triiodio-L-thyronine (T3), mixtures thereof (e.g., Thyroid Extract, USP or Liotrix, a 4:1 mixture of T4:T3), or a compound which mimics the activity of thyroid hormone or otherwise stimulates the signaling pathway of the thyroid receptor, either by interacting with the thyroid receptor or with downstream signaling pathway elements.

The term "thyroid axis antagonist" refers to a compound which acts to decrease thyroid hormone activity in a subject. Thyroid axis antagonists include 6-n-propyl-2- thiouracil (propylthiouracil or PTU), methimazole, carbimazole, and other compounds known to the art to reduce thyrotropic hormones, thyroid hormones, or thyroid receptor signaling.

The term "TRH analog" refers to compounds which have the activity of stimulating the release of thyroid hormone from the thyroid gland. TRH analogs generally have all or part of the structural characteristics of the peptide L-pyroglutamyl-L-histidyl-L-proline amide. TRH analogs for use in the methods of the instant invention include those TRH analogs disclosed in U.S. Patent No. 4,426,378, such as MK771 (pyro-2-aminoadipyl- histidyl-thiazolidine-4-carboxamide), histidyl-proline diketopiperazine, L-2-ketopiperidine- 6-carbonyl-L-histidyl-L-thiazolidine-4-carbonyl-B-alaninamide, tetrapeptide amide L- pyroglutamyl-L-histidyl-L-prolyl-B-alaninamide, L-2-oxo-oxazolidine-4-carboxylic acid,

L-2-oxo-oxazolidine-4-carbonyl-L-histidyl-L-prolineamide, L-trans-5-methyl-2-oxo- oxazolidine-4-carbonyl-histidyl-L-prolineamide, L-2-oxothiaxolidine-4-carbonyl,-L- histidyl-L-prolineamide, 3-oxo-5-carboxyperhydro-l ,4-thiazine-histidine-proline-NH₂, 3- oxo-5-carboxyperhydro-l ,4-thiazine-histidine-pipecolic acid-NH₂, 3-oxo-5- carboxyperhydro-1 ,4-thiazine-histidine-proline-HNCH₂CH₂CH₂CH₃, 3-oxo~5- carboxyperhydro- 1 ,4-thiazine-histidine-proline-NHCH₂CH₂C₆H₅, γ-carboxy-γ- butyrolactone-histidine-proline-NHCH₂CH₂C₆H₅, γ-carboxy-γ-butyrolactone-histidine- proline-NH₂, γ-carboxy-γ-butyrolactone-histidine-pipcolic acid-NH2, γ-carboxy-γ- butyrolactone-histidine-proline-NH-CH₃, γ-carboxy-γ-butyrolactone-histidine-proline- NHCH₂CH₂CH₂CH₃, γ-carboxy-γ-butyrolactone-histidine-proline-NHCH₂CH₂C₆H₅, pharmaceutically acceptable salts thereof, and the like.

The methods disclosed herein provide for the treatment of non-thyroid disorders which respond to IGF. In accordance with the instant methods, IGF, preferably IGF-I, and a thyroid axis agonist are administered to a subject suffering from a non-thyroid disorder which responds to IGF, thereby alleviating the symptoms of the disorder. In a particularly preferred embodiment, the IGF-I is administered as a complex with IGFBP-3. Further disclosed are methods for treatment of non-thyroid disorders which respond to IGFBP-3. In accordance with these methods, IGFBP-3 and a thyroid axis antagonist are administered to a subject, thereby alleviating the symptoms of the disorder. In a preferred embodiment, the IGFBP-3 is administered as a complex with mutant IGF. Also disclosed herein are methods for treating non-thyroid disorders associated with an excess of IGF. In accordance with the instant methods, thyroid axis antagonists are administered to subjects suffering from non-thyroid disorders associated with an excess of IGF, thereby alleviating the symptoms of the disorder.

In methods involving the administration of IGF, IGF-I is preferred, and species- matched IGF-I (i.e., IGF-I from the same species as the subject to which the IGF-I is to be administered) is more preferred. A particularly preferred form of IGF-I is a complex of IGF-I and IGFBP-3, more preferably species-matched IGF-I and IGFBP-3. Where IGF- I/IGFBP-3 is administered, the complex is preferably 1 :3 to 3:1 on a molar basis, more preferably 1.5:1 to 1 :1.5, most preferably 1 :1. Preferably the IGF-I and IGFBP-3 are complexed prior to administration, which can be easily accomplished by methods well known in the art. Preferably, the complex is formed by mixing approximately equimolar amounts of IGF-I and IGFBP-3 dissolved in physiologically compatible carriers such as normal saline, or phosphate buffered saline solution. More preferably, a concentrated solution of rhIGF-I and a concentrated solution of rhIGFBP-3 are mixed together for a sufficient time to form an equimolar complex. Most preferably, rhIGF-I and rhIGFBP-3 are combined to form a complex during purification, as described in International Patent Application No. WO 96/40736.

For methods involving the administration of IGFBP-3, the IGFBP-3 is preferably species-matched. More preferably, the IGFBP-3 is administered as a complex with mutant IGF-I. The mutant IGF-I is preferably also species-matched (i.e., the backbone of the IGF-

I, exclusive of the amino acids where mutations are present, is the same amino acid sequence as the corresponding positions of wild-type IGF-I from the same species as the subject). Where a complex of mutant IGF-I and IGFBP-3 is administered, the complex is preferably 1 : 3 to 3 : 1 on a molar basis, more preferably 1.5 : 1 to 1 : 1.5 , most preferably 1 :1. Preferably the mutant IGF-I and IGFBP-3 are complexed prior to administration, which can be easily accomplished by methods well known in the art. Preferably, the complex is formed by mixing approximately equimolar amounts of mutant IGF-I and IGFBP-3 dissolved in physiologically compatible carriers such as normal saline, or phosphate buffered saline solution. More preferably, a concentrated solution of mutant rIGF-I and a concentrated solution of rhIGFBP-3 are mixed together for a sufficient time to form an equimolar complex. Most preferably, mutant rIGF-I and rhIGFBP-3 are combined to form a complex during purification.

In methods involving the administration of IGF, IGFBP-3, IGF/IGFBP-3 complex, or mutant IGF-I/IGFBP-3 complex, the IGF, IGFBP-3, IGF/IGFBP-3 complex, or mutant IGF-I/IGFBP-3 complex is preferably administered by a parenteral route, including, but not limited to, intravenous (IV), intramuscular (IM), subcutaneous (SC), intraperitoneal (IP), intranasal, and inhalant routes. IV, IM, SC, and IP administration may be by bolus or infusion, and in the case of SC, may also be by slow release implantable device, including, but not limited to pumps, slow release formulations, and mechanical devices.

For parenteral administration, compositions of IGF, IGFBP-3, IGF/IGFBP-3 complex, or mutant IGF-I/IGFBP-3 complex may be semi-solid or liquid preparations, such as liquids, suspensions, and the like. Preferably the IGF, IGFBP-3, IGF/IGFBP-3 complex, or mutant IGF-I/IGFBP-3 complex is formulated for administration in a liquid formulation comprising additional components such as physiologically compatible carriers and/or pharmaceutically acceptable excipients as are known in the art. Acceptable additional components include salts, buffers, antimicrobial agents, buffers, bulking agents, osmolytes, antioxidants, detergents, surfactants, and the like. Preferably, the IGF, IGFBP-3, IGF/IGFBP-3 complex, or mutant IGF-I/IGFBP-3 complex for parenteral administration is IGF, IGFBP-3, IGF/IGFBP-3 complex, or mutant IGF-I/IGFBP-3 complex in an isotonic solution of mannitol and sucrose (3:2 molar ratio), as disclosed in copending U.S. Patent Application No. 09/089,062.

In methods requiring the administration of thyroid axis agonists, the dose of the thyroid axis agonist must normally be adjusted for the individual subject, as is well known in the art. Induction of frank hyperthyroidism is generally not required, and for some indications should be avoided. For example, induction of hyperthyroidism may be tolerated where the symptoms of hyperthyroidism do not confound or otherwise reduce the treatment effect (e.g., in the treatment of renal indications). Conversely, as will be understood by one of skill in the art, induction of hyperthyroidism is generally contraindicated where the symptoms of hyperthyroidism would confound or otherwise reduce the treatment effect (e.g., in the treatment of catabolic conditions). Normally, the treatment of the subject will initiated at a low to intermediate dose, and the patient will be observed for the onset of hyperthyroidism, although patients with a history of cardiac disease are generally started with a lower initial dose, as is known in the art. Hyperthyroidism may be recognized by its well known symptoms, which include nervousness, emotional liability, sleeplessness, tremors, excessive sweating, heat intolerance, weight loss and loss of strength. If symptoms of hyperthyroidism occur and induction of hyperthyroidism is contraindicated, then the dose of the thyroid axis agonist should be reduced, and the patient should be further observed for continued symptoms of hyperthyroidism. If the hyperthyroidism symptoms do not resolve, the dose of the thyroid axis agonist should be further reduced until the symptoms of hyperthyroidism resolve.

Thyroid axis agonists are available in a variety of formulations, including parenteral and oral dosage forms. Oral formulations are preferred, but parenteral formulations are also acceptable, and may be more convenient in an in-patient setting. The dosage of thyroid axis agonist must also be adjusted according to the identity of the thyroid axis agonist which is administered with the IGF, IGF-I of IGF-I/IGFBP-3 complex, as is known in the art. For example, where the thyroid axis agonist is T4, the daily dose is about 25 to 250 μg/day, more preferably 50 to 200 μg/day. As is known in the art, T4 administration may be initiated with a higher "loading" dose, ranging from 50 to 500 μg/day. If T3 is the thyroid axis agonist, then the doses are normally lower, not exceeding 100 μg/day, more preferably from about 10 to 100 μg/day. Where the thyroid axis agonist is iodinated casein, it is preferably administered orally at 50 to 1000 mg/day. For TRH and TRH analogs, the dosage is preferably in the range of about 1 μg to 20 mg/kg of body weight.

Similarly for methods requiring the administration of thyroid axis antagonists, the dose of the thyroid axis antagonist is also normally titrated for the individual subject, as is well known in the art. Induction of frank hypothyroidism is not required, although it may be advantageous is some cases for the proper working of the invention. Similar to the procedure for thyroid axis agonists, thyroid axis antagonists are administered at an intermediate dose, and the patient observed for the onset of hypothyroidism. Hypothyroidism may be recognized by its well known symptoms, including (in adults) lethargy, constipation, cold intolerance, menorrhagia (in women of reproductive age), reduced intellectual and motor activity, dry hair, dry skin, muscle aches, reduced auditory acuity, and deepening and hoarsening of the voice. In extreme cases, florid myxedema may be present, as indicated by a dull, expressionless face, sparse hair, periorbital puffiness, enlarged tongue and pale, cool skin which feels rough and doughy. The thyroid antagonist dose should be reduced if florid myxedema appears, to avoid the possibility of myxedema coma, a serious and frequently fatal condition. Upon the appearance of the signs of hypothyroidism which fall short of florid myxedema, the dose of the thyroid axis antagonist may be reduced to the point at which the symptoms of hypothyroidism resolve, as will be understood by one of skill in the art.

Thyroid axis agonists may be produced in a variety of different formulations, including parenteral and oral dosage forms. Oral formulations are preferred, but parenteral formulations are also acceptable, and may be more convenient in an in-patient setting. The dosage of thyroid axis agonist must also be adjusted according to the identity, formulation and route of administration of the thyroid axis agonist which is administered with the IGF, IGF-I of IGF-I/IGFBP-3 complex, as is known in the art. Where the thyroid axis antagonist is propylthiouracil, the dose of propylthiouracil may be from 1 to 400 mg/day. A subject is normally initiated with a dose of 50 to 400 mg/day, typically divided into three equal doses, and maintained at 50 to 100 mg/day divided into two or three equal doses. For methimazole and carbimazole, the dose may be from 0.1 to 50 mg/day. Typically, a subject is initiated with 5 to 50 mg/day, and maintained on 1 to 5 mg/day.

Thyroid axis agonists and thyroid axis antagonists may be formulated in any manner known to the art. Formulations for parenteral administration are generally formulated as liquids, but may also be in gel or solid depot form. Formulations for oral administration are generally in tablet or capsule form, although syrups and liquids are also acceptable. Formulations of thyroid axis agonists and thyroid axis antagonists generally include excipients, such as salts, buffers, bulking agents, detergents, binding agents, surfactants, stabilizers, preservatives, anti-oxidants, lubricants, coating agents, and other pharmaceutically acceptable excipients as are known in the art. For methods in which (a) a thyroid axis agonist and IGF or (b) a thyroid axis antagonist and IGFBP-3 are administered, the administration of the two compounds may be simultaneous, overlapping, or separated in time, as long as the subject experiences exposure to both compounds at the same time. Where the two compounds are formulated for the same route and schedule of administration, the administration is preferably simultaneous or nearly simultaneous (e.g., concurrent or serial injections). However, in some embodiments, the routes and schedules of administration for the two compounds will be different, making simultaneous administration infeasible. A subject will be considered to have been administered either (a) a thyroid axis agonist and IGF or (b) a thyroid axis antagonist and IGFBP-3 if the subject experiences simultaneous systemic exposure to both compounds, regardless of when the compounds were administered.

The patents, patent applications, and publications cited throughout the disclosure are incoφorated herein by reference in their entirety.

The present invention has been detailed both by direct description. Equivalents and modifications of the present invention will be apparent to those skilled in the art, and are encompassed within the scope of the invention.

Claims

1. A method for alleviating symptoms of a non-thyroid disorder which responds to insulin-like growth factor (IGF), comprising: administering an effective amount of IGF to a patient suffering from said disorder; and administering an effective amount of a thyroid axis agonist to said subject, thereby alleviating symptoms of said disorder.

2. The method of claim 1 wherein said IGF is IGF-I.

3. The method of claim 2 wherein said IGF-I is human IGF-I.

4. The method of claim 3wherein said IGF-I is complexed with insulin-like growth factor binding protein 3 (IGFBP-3).

5. The method of claim 4 wherein said IGFBP-3 is human IGFBP-3.

6. The method of claim 5 wherein said IGFBP-3 is native human IGFBP-3.

7. The method of claim 2 wherein said IGF-I is complexed with insulin-like growth factor binding protein 3 (IGFBP-3).

8. The method of claim 8 wherein said IGFBP-3 is human IGFBP-3.

9. The method of 9 wherein said human IGFBP-3 is native human IGFBP-3.

10. The method of claim 1 wherein said thyroid axis agonist is selected from the group consisting of L-thyroxine (T4), L-thyronine (T3), thyroid stimulating hormone (TSH), thyrotropin releasing hormone (TRH), and TRH analogues.

11. A method for alleviating symptoms of a non-thyroid disorder which responds to insulin-like growth factor binding protein (IGFBP-3), comprising: administering an effective amount of IGFBP-3 to a subject suffering from said disorder; and administering an effective amount of a thyroid axis antagonist, thereby alleviating symptoms of said disorder.

12. The method of claim 11 wherein said IGFBP-3 is human IGFBP-3.

13. The method of claim 12 wherein said human IGFBP-3 is native human IGFBP- 3.

14. The method of claim 11 wherein said IGFBP-3 is complexed with mutant IGF-I.

15. The method of claim 12 wherein said mutant IGF-I is mutant human IGF-I.

16. The method of claim 12 wherein said mutant IGF-I comprises an alteration in amino acid sequence at a position selected from the group consisting of 24, 31, 49, 50, 51, 53, 55, 56, and 60.

17. A method for alleviating symptoms of an insulin-like growth factor (IGF) - dependent disorder which is not a thyroid disorder, comprising: administering an effective amount of a thyroid axis antagonist to a subject suffering from said disorder, thereby alleviating symptoms of said disorder.

18. The method of claim 17, wherein said thyroid axis antagonist is selected from the group consisting of propylthiouracil, methimazole and carbimazole.