HK1148194A - Adrenocorticotropic hormone analogs and related methods - Google Patents

Description

Corticotropin analogs and related methods

The present application is a divisional application of chinese patent application 200580042427.7(PCT/US2005/038789) entitled "corticotropin analogs and related methods" filed on 25/10/2005.

Government support

The subject of the present application was supported by the research Foundation (Foundation No. NIH: DK50870) of the National Institutes of Health and the Foundation (Foundation No. NSF IBN-0132210) of the National Science Foundation. The government therefore has certain rights in the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

The benefit of U.S. provisional patent application serial No. 60/622,436 entitled "composition and method composition for treating premature labor, cushing's syndrome, and related conditions," filed on month 10, 27 of 2004, which is hereby incorporated by reference.

Technical Field

The present invention relates to ACTH analog compounds and related pharmaceutical compositions and methods of treatment.

Background

Corticotropin (corticotropin), also known as adrenocorticotropic hormone (ACTH), is a major hormone secreted by the pituitary gland, which is thought to be a mediator in the production of a variety of important growth and physiological control steroids. ACTH stimulates the adrenal cortex. More specifically, it stimulates secretion of glucocorticoids such as cortisol in humans (or corticosterone in rodents), and rarely controls secretion of the other major steroid hormone aldosterone from the adrenal cortex. ACTH binds to the MC-2R corticotropin receptor expressed in the adrenal gland.

ACTH is secreted from the anterior pituitary in response to corticotropin-releasing hormone (CRH) originating from the hypothalamus. In the pituitary gland, ACTH is derived from the large precursor molecule Proopiomelanocortin (POMC), which is cleaved by specific peptidases. The effects of ACTH on steroid synthesis include increased cholesterol esterase, cholesterol transport to and across the mitochondrial membrane, binding of cholesterol to P450SCC, and thus increased pregnenolone production (see Nussey, S. and S. Whitehead, Endocrinology: An Integrated Approach, BIOS Scientific Publishers Ltd. (2001)). Subsequent effects include induction of steroidogenic enzymes and significant structural changes characterized by hypervascularization, cell overgrowth and hyperplasia. This is particularly evident after a prolonged period of excess secretion of ACTH.

The steroid glucocorticoids are produced by zona fascicularis-reticula cells (fascicula-reticula cells) of the adrenal gland and are secreted in response to increased plasma adrenocorticotropic hormone (ACTH) levels. Glucocorticoids are involved in sugar, protein and fat metabolism and have been shown to have anti-inflammatory properties and to be over-secreted during stress. Excess glucocorticoids damage the hippocampus of the limbic system of the brain, which is critical for cognitive functions such as learning and memory. See, e.g., saplsky, r.m., ann.n.y.acad.sci.746: 294 (1994); and McEwen, b.s., ann.n.y.acad.sci.746: 134(1994). In addition, the neurotoxicity and neurovirulence of glucocorticoids play an important role in the development and aging of the nervous system and in neurological disorders associated with hippocampal damage. See, e.g., deKloet, e.r. et al, ann.n.y.acad.sci.746: 8(1994).

Corticosteroids are steroid hormones structurally related to cholesterol. These hormones are synthesized in the adrenal cortex and include glucocorticoids (e.g., corticosteroids), mineralocorticoids (e.g., aldosterone), and weak androgens and estrogens. Like the thyroid gland, the function of the adrenal gland is under the control of the Hypothalamus (HPT) and pituitary gland (PIT). When corticosteroid (naturally occurring glucocorticoid) levels fall to a set point, the hypothalamus releases CRH (corticotropin releasing hormone), which stimulates adrenocorticotropic hormone (ACTH) release from the pituitary. ACTH is a prohormone that stimulates the synthesis and secretion of corticosteroids (which have minimal effect on aldosterone synthesis/secretion) and the growth of the adrenal glands.

There is a need for compounds that bind to ACTH receptors but have reduced activation of corticosteroid secretion, for example, in the treatment of ACTH-related disorders, including cushing's syndrome, impaired immune response due to excessive corticosteroid secretion, and premature labor for certain adrenal-related reasons.

Cushing's syndrome is a condition resulting from increased secretion of corticosteroids from the adrenal cortex. Adrenocortical hyperactivity may be ACTH dependent or independent of ACTH regulation, such as corticosteroid production resulting from adrenocortical adenoma carcinoma. A common cause of cushing's syndrome is the overproduction of ACTH by the pituitary gland. This increase in ACTH levels in the bloodstream is generally due to pituitary adenoma (cushing's disease), but in rare cases the cause is different. Cushing's syndrome, which arises from the extra-pituitary ACTH site, is called ectopic cushing's syndrome. Examples of ectopic sites include thymoma, medullary thyroid carcinoma, pheochromocytoma, islet cell tumor of pancreas, and oat cell cancer of lung. However, the vast majority of the causes of cushing's syndrome in humans are pituitary adenomas. Symptoms of cushing's syndrome include increased body weight, central obesity, hypersecretion of steroids, increased excretion of urothelial alcohol, shiny face, weakness, fatigue, back pain, headache, impotence, altered mental status, muscle atrophy, increased thirst and increased urine compared to a mammal not suffering from the disease. The Diagnosis and treatment of Cushing's syndrome remains a challenge (see Oldfield, E.W. et al, N.Engl. J.Med., 325: 897-905 (1991); Findling, J.W. et al, "Diagnosis and differentiation Diagnosis of curing's syndrome," Endocrinol. Metab. Clin. North Am., 30: 729-47 (1995); Orth, D.N., "curing's syndrome," N Engl J. Med., 332: 791-803 (1995)). There is currently no medical treatment for cushing's syndrome. In the center of the experienced discipline, the overall cure rate for surgically resected ACTH-secreting pituitary microadenomas is about 70-80%, but for large adenomas, the cure rate is only about 30%, and the need for extensive surgical resection predicts a significant risk for the surrounding normal pituitary tissue, leading to partial or complete hypopituitarism in about 80% of the pathologies (Simmons, N.E. et al, "Serum Cortisol response to renal tissue for treating disease," J.Neurosurg., 95: 1-8 (2001); Mampalam, T.J. et al, "venous microbial surgery for treating's disease: A report of 216cases, Medium. n.Med., 109: 487-93 (1988); and Transiner, P.J. et al," respiration therapy "diagnosis" 73: 1-7). Thus, there remains a need for effective therapies for ACTH from cushing's syndrome, which is either a sporadic pituitary tumor or an ectopic source, without risk to the patient. Compounds that bind ACTH receptors but have reduced activation of cortisol secretion may also be useful in treating the hypothalamic-pituitary-adrenal axis that causes premature labor. In all births, preterm birth is present in about 7-10% and accounts for a relatively large proportion of perinatal morbidity and mortality (McCormick, m.c., "The balance of low birth weight to injury mortality and childhood mortality," N Engl J med., 312: 82-90 (1985)). For a variety of reasons, there is a need to prevent spontaneous miscarriage and premature birth as well as to prolong pregnancy in women. There is a need to prolong pregnancy for the following reasons: (i) more likely to deliver a live baby; (ii) reducing the incidence of health complications in premature infants; and (iii) reduce the period of time that the premature infant receives special care (which must be received due to its size and viability, even if healthy). All of the following factors (i) survival at birth, (ii) infant health, and (iii) the ability of an infant to be discharged from a hospital in a timely manner under the care of parents, affect the well-being and well-being of parents and relatives. Premature delivery also has social implications, including a very large socio-economic impact from the care of prematurely born infants.

In agriculture and aquaculture, it is more cost effective when organisms are raised at high population densities. In mammals, poultry and fish, however, this often results in an overproduction of adrenal stress hormones, which can have deleterious consequences including impaired immune function and reduced growth. It is also desirable to reduce the level of adrenal stress hormones in these situations or other situations that result from prolonged stress and lead to adverse health changes (e.g., reduced immune function and susceptibility to disease).

Various compositions and methods are useful for reducing ACTH levels, for example, via certain receptors for Arginine Vasopressin (AVP). U.S. patent No. 6,380,155, filed 5/3/2000, relates to the use of certain vasopressin receptor antagonist compositions for modulating ACTH release. Compositions that modulate ACTH levels to treat ACTH-related disorders are desirable, for example, compounds that bind to the ACTH MC-2R receptor but simultaneously reduce and eliminate ACTH-induced corticosteroid production such that adverse conditions associated with elevated ACTH levels are alleviated.

Brief description of the invention

Modified adrenocorticotropic hormone (ACTH) peptides ("ACTH analogs") are provided, which reduce and eliminate ACTH-induced corticosteroid secretion compared to unmodified ACTH. Preferably, ACTH analogs also reduce corticosteroid secretion by adrenal membranes in the presence of unmodified ACTH.

ACTH analog compounds preferably contain at least amino acids 1-24 of unmodified ACTH with one or more amino acid substitutions. ACTH analog compounds may include one or more amino acid substitutions or be truncated relative to the unmodified ACTH amino acid sequence. Unmodified human ACTH is a 39 amino acid residue polypeptide having the sequence: N-Ser¹-Tyr-Ser-Met-Glu⁵-His-Phe-Arg-Trp-Gly¹⁰-Lys-Pro-Val-Gly-Lys¹⁵-Lys-Arg-Arg-Pro-Val²⁰-Lys-Val-Tyr-Pro-Asn²⁵-Gly-Ala-Glu-Asp-Glu³⁰-Ser-Ala-Glu-Ala-Phe³⁵-Pro-Leu-Glu-Phe³⁹-Ac (SEQ ID NO.1), wherein N and Ac represent the amino and carboxy termini of the molecule, respectively. ACTH analogs may also include compounds that include the sequence N-Ser¹-Tyr-Ser-Met-Glu⁵-His-Phe-Arg-Trp-Gly¹⁰-Lys-Pro-Val-Gly-Lys¹⁵-Lys-Arg-Arg-Pro-Val²⁰-Lys-Val-Tyr-Pro-Ac (SEQ ID NO: 2) having one or more substituted and modified peptides, wherein N and Ac represents the amino and carboxy termini of the molecule, respectively. Preferred ACTH analog compounds include at least amino acid residues 1-19 of at least hACTH and mACTH, more preferably amino acid residues 1-24 of hACTH or mACTH, with one or more amino acid substitutions.

ACTH analog compounds include polypeptides comprising the ACTH sequence of SEQ ID NO: 1 or SEQ ID NO: 2, which modification results in one or more of the following preferred ACTH analog biological functions: (1) decreased secretion of corticosteroids by the adrenal membrane in the presence of ACTH analogs as compared to unmodified ACTH, (2) decreased secretion of corticosteroids by the adrenal membrane in the presence of endogenous ACTH, and (3) increased binding affinity for MC-2R with decreased activation of MC-2R receptors as compared to unmodified ACTH. Examples of preferred ACTH amino acid substitutions to form ACTH analog compounds include one or more of the following: (1) retaining the amino acids at positions 6-9 and/or facilitating or retaining MC-2R binding by one or more amino acid residue substitutions at positions 1-13, (2) one or more amino acid residue substitutions at positions 25-18 to prevent or counter enzymatic cleavage at positions 15-18, (3) one or more amino acid residue substitutions at positions 15-18 to render the ACTH analog free of adjacent basic side chain-bearing amino acid residues at positions 15-18, (4) one or more amino acid residue substitutions or truncations at positions 20-24 to increase the serum half-life of the ACTH analog, (5) reducing corticosteroid secretion by the adrenal membrane in the presence of the ACTH analog compound as compared to the unmodified ACTH peptide, and one or more amino acid residue substitutions at positions 20-36, (6) one or more amino acid residue substitutions or preferred truncations at positions 25-39 to provide a desired release profile for the ACTH analog compound, or (7) truncating amino acid residues 25-39 such that the amino acid residue at position 24 forms the carboxy terminus of the molecule.

ACTH analog compounds preferably bind to ACTH receptors, such as the melanocortin 2 receptor (MC-2R) in the adrenal membrane. More preferably, ACTH analog compounds do not or weakly activate MC-2R expressing cells and inhibit or reduce the effects of unmodified endogenous ACTH.

In a first embodiment, the plurality of ACTH analog compounds can include one or more amino acid substitutions or truncations relative to an unmodified human ACTH amino acid sequence. For example, a composition comprising an isolated ACTH analog peptide can include SEQ ID NO: 2, a peptide:

seq ID NO: 2 a Pro residue at position 19 is substituted with the amino acid Trp; or

b. Selected from the group consisting of SEQ ID NO: 2 such that amino acid residues 16, 17 and 18 of the ACTH analog do not include any two adjacent amino acid residues selected from Lys and Arg; and in SEQ ID NO: 2 is selected from the group consisting of Lys, Arg, Gln, Gly, Ala, Val, Leu, Ile, and amino acid analogs having an alkyl side chain (e.g., Nle). Optionally, the ACTH analog peptide may include at least one Ala, Gly, or another amino acid with an alkyl side chain (i.e., Val, Leu, Ile, amino acid analogs containing an alkyl side chain such as Nle) and at least one Arg residue, which are set forth in SEQ ID NO: 2 at any two of amino acid positions 15, 16, 17 or 18. Optionally, the ACTH analog consists essentially of SEQ id no: 2 sequence composition: SEQ ID NO: 2 the Pro residue at position 19 is replaced with the amino acid Trp; SEQ ID NO: 2 the amino acid at position 15 is selected from Lys, Ala and Gln; and the ACTH analog peptide may be represented in a sequence selected from SEQ ID NOs: 2, such that amino acid residues 16, 17, and 18 of the ACTH analog do not include any two adjacent amino acid residues selected from Lys and Arg. Preferably, the ACTH analog peptide includes at amino acid residues 6, 7,8, and 9 the amino acid sequence of SEQ ID NO: 4. ACTH analog peptides may also include SEQ ID NOs: 12.

The first embodiment further comprises a polypeptide having the amino acid sequence of SEQ ID NO: 1, such as the ACTH analog of the peptide of SEQ ID NO: 1, amino acid residue substitution at position 19, 26, 30 or 36. ACTH analog peptides also include SEQ ID NOs: 1 peptide:

seq ID NO: 1, a Pro residue at position 19 substituted with the amino acid Trp; or

b. Selected from the group consisting of SEQ ID NO: 1 such that amino acid residues 16, 17 and 18 of the ACTH analog do not include any two adjacent amino acid residues selected from Lys and Arg; and in SEQ ID NO: 1 is selected from the group consisting of Lys, Arg, Gln, Gly, Ala, Val, Leu, Ile, and Nie (or another amino acid analog having an alkyl side chain).

Other preferred ACTH analog peptides include the sequence set forth for SEQ ID NO: 1 or SEQ ID NO: 2 such that the ACTH analog peptide includes at amino acid residues 15-19 the amino acid sequence of SEQ id no: 6. ACTH analog peptides also include truncations of one or more of the peptides. ACTH analogs also include the amino acid sequence of SEQ ID NO: 1 amino acid residues 25-39 truncation. ACTH analog peptide SEQ ID NO: 20 is particularly preferred.

In some embodiments, the treatment is performed in vitro as determined by Serum Corticosteroid Induction Assay (Serum Corticosteroid Induction Assay) in combination with administration of SEQ ID NO: 2, administration of the ACTH analog peptide reduces corticosterone production induced by ACTH in the adrenal membrane by at least 10%, preferably by up to 100%. For example, in a second embodiment, ACTH analogs are modified ACTH peptides that reduce corticosteroid secretion by adrenal membranes in the presence of the ACTH analog compared to unmodified ACTH.

In some embodiments, administration of the ACTH analog peptide reduces ACTH-induced Corticosteroid secretion by at least 10%, as determined by an in vitro Serum Corticosteroid Inhibition Assay (Serum Corticosteroid Inhibition Assay). In a third embodiment, ACTH analogs are modified ACTH peptides that reduce corticosteroid secretion by adrenal membrane in the presence of endogenous ACTH.

In some embodiments, compositions are provided that include an isolated nucleic acid sequence of SEQ ID NO: 2, wherein the ACTH analog peptide binds to the adrenal membrane and replaces the peptide of SEQ ID NO: 2. Peptide Binding can be determined by in vitro Serum-Free Adrenal Competitive Binding Assay (Serum-Free additional Binding Assay). For example, in a fourth embodiment, ACTH analogs are modified ACTH peptides that bind to adrenal ACTH receptors, such as the MC-2R receptor, and preferably have increased MC-2R binding affinity and reduced activation of the MC-2R receptor (as compared to unmodified ACTH). Preferably, the ACTH analog peptide can bind to and replace SEQ ID NO: 2, wherein the binding of the peptide can be determined by an in vitro serum-free adrenal competition binding assay. Most preferably, the ACTH analog peptide is expressed as a sequence that is more than SEQ ID NO: 2 binds to MC-2R adrenal membrane with at least 2-fold greater affinity.

In some embodiments, ACTH analog peptides reduce ACTH-induced corticosterone production of the Adrenal membrane as determined by an in vitro Serum-free Adrenal Inhibition Assay (Serum-free additive Inhibition Assay). For example, in a fifth embodiment, ACTH analog compounds can reduce corticosteroid induction by unmodified ACTH in explant tissue in vitro. ACTH analogs include peptides that reduce ACTH-induced corticosterone production by adrenal membranes in an in vitro serum-free adrenal suppression assay.

In a sixth embodiment, extended half-life ACTH analogs are provided. The extended half-life ACTH analog is identified as having a first activity measured by serum corticosteroid concentration measured in vivo that is greater than a second activity measured by serum-free concentration of corticosteroid measured by in vitro activity, wherein the in vivo activity is determined by the serum adrenal corticosteroid inhibition assay of example 2 and the in vitro activity is determined by the in vitro serum-free adrenal corticosteroid inhibition assay of example 4.

In a seventh embodiment, methods are provided for screening for ACTH analogs that can block excess ACTH while maintaining adrenal health. A variety of ACTH analogs can be prepared and administered to patients to assess the induction of cortisone in vivo.

In an eighth embodiment, the disclosure relates to pharmaceutical compositions comprising ACTH analogs and administering to a subject in a manner suitable for treating symptoms associated with ACTH-related disorders. The ACTH analogs described herein can be added to pharmaceutical compositions to treat ACTH-related disorders, such as ACTH overexpression in a human or animal. Methods of producing ACTH analogs and related pharmaceutical compositions comprising ACTH analogs are also provided. For example, in one aspect, methods are provided for screening a class of ACTH analogs for compounds that can be used to block excess ACTH while maintaining adrenal health.

ACTH analog compounds are useful for treating diseases associated with ACTH levels, such as diseases that respond to modulation of ACTH receptors (e.g., MC-2R). Also provided are compounds useful for modulating corticosteroid secretion or corticosteroid levels. ACTH analog compounds can be administered to treat disorders associated with modulation of ACTH levels, such as reducing the effects of high levels of ACTH in a patient while maintaining the health of adrenal function. For example, ACTH analog compositions are useful for treating ACTH-related disorders such as cushing's syndrome, impaired immune response due to excessive corticosteroid secretion, causing premature labor (e.g., through the hypothalamic-pituitary-adrenal axis), and related disorders. In one aspect, a plurality of ACTH analogs are prepared and administered to a patient to assess induction of cortisone in vivo. In another aspect, methods of treating a veterinary subject, such as methods of reducing stress hormones to facilitate healthy growth of agricultural and aquaculture species at high population densities, are provided.

Brief Description of Drawings

In the drawings:

figure 1 shows corticosterone levels measured in vivo following injection of various ACTH analog compounds compared to corticosterone levels measured following administration of unmodified ACTH.

Figure 2 compares corticosterone induced in vivo by administration of unmodified ACTH, combined ACTH analog administration with unmodified ACTH, administration of ACTH analog followed by unmodified ACTH alone, and ACTH analog alone.

Figure 3 is a graph comparing results of assays of competitive binding of unmodified ACTH and ACTH analog compounds to adrenal ACTH receptors.

Figure 4 compares the induction of corticosterone in an extradermal adrenal membrane in vitro by unmodified ACTH, ACTH analogs in combination with unmodified ACTH, and ACTH analogs alone.

Figure 5 compares the in vivo and in vitro corticosterone-inducing activity of different ACTH agonist compounds compared to the unmodified ACTH activity (i.e., 100%).

Detailed Description

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In case of conflict, the present specification, including definitions, will control. Preferred methods and materials are described below, but methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. All publications, patent applications, patent documents, and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

As used herein, the term "unmodified adrenocorticotropic hormone" ("unmodified ACTH") means a peptide hormone produced by the anterior pituitary gland that stimulates the adrenal cortex to secrete glucocorticoids that help cells synthesize glucose by gluconeogenesis, catabolize proteins, mobilize free fatty acids, and suppress inflammation in allergies. One such hormone is corticosteroids, which regulate sugar, fat and protein metabolism.

As used herein, "corticosteroid" includes the human corticosteroid cortisol and the rodent corticosteroid corticosterone. The term "about" as used in reference to a quantity includes variations from the stated quantity that are equivalent to the stated quantity, e.g., a quantity that does not substantially differ from the stated quantity for the intended purpose or function.

The nomenclature "P (x-y)" (wherein P is the name of the polypeptide and x and y are integers) as used herein refers to an amino acid sequence consisting of consecutive amino acids from position (x) to position (y) of the polypeptide referred to as "P". For example, "hACTH (1-24)" refers to a polypeptide of 24 contiguous amino acids consisting of residues 1 to 24 from the amino terminus of the human ACTH peptide. "mACTH" refers to murine ACTH. In particular, hACTH (1-24) and mACTH (1-24) are the same peptide sequence.

The nomenclature "aXb" (where a and b are the single letter abbreviations for amino acids, and X is a number) means that the amino acid "a" at the "X" position in the unmodified ACTH peptide is replaced with the amino acid "b". For example "(V26F, E30K) mACTH" refers to a 39 amino acid mouse ACTH molecule in which Val at position 26 is replaced with Phe and Glu at position 30 is replaced with Lys from the amino terminus of the molecule. Similarly, the nomenclature "α β χ X-Y δ ∈ Φ" (where α, β, χ, δ, ε, and Φ represent the single letter abbreviations for amino acids, and X and Y are numbers) means that the consecutive amino acids "α β χ" at positions X to Y are replaced by the amino acid "δ ∈.

ACTH analog compounds include compounds that include an ACTH sequence having one or more structural modifications that provide one or more of the following preferred biological functions of ACTH analogs (1) reduced corticosteroid secretion by the adrenal membrane in the presence of the ACTH analog as compared to unmodified ACTH, (2) reduced corticosteroid secretion by the adrenal membrane in the presence of endogenous ACTH, and (3) increased MC-2R binding affinity with reduced activation of the MC-2R receptor as compared to unmodified ACTH. ACTH analog compounds are thought to act by binding to the melanocortin 2 receptor (MC-2R), weakly activating MC-2R expressing cells, and blocking the action of endogenous ACTH on the MC-2R receptor. Examples of preferred ACTH amino acid substitutions for forming ACTH analog compounds with one or more desired functions include: (1) retaining the amino acids at positions 6-9 and/or facilitating or retaining MC-2R binding by one or more amino acid residue substitutions at positions 1-13, (2) one or more amino acid residue substitutions at positions 25-18 to prevent or counter enzymatic cleavage at positions 15-18, (3) one or more amino acid residue substitutions at positions 15-18 to avoid the presence of two adjacent basic amino acids, (4) one or more amino acid residue substitutions or truncations at positions 20-24 to increase the serum half-life of the ACTH analog, (5) one or more amino acid residue substitutions or preferably truncations at positions 25-39 to provide the desired release characteristics to the ACTH analog compound, or (6) truncating amino acid residues 25-39, the amino acid residue at position 24 forms the carboxy terminus of the molecule.

ACTH analog compounds

In a first embodiment, the plurality of ACTH analog compounds can include one or more amino acid substitutions or truncations relative to an unmodified human ACTH amino acid sequence. Unmodified human ACTH is a 39 amino acid residue polypeptide having the sequence: N-Ser¹-Tyr-Ser-Met-Glu⁵-His-Phe-Arg-Trp-Gly¹⁰Lys-Pro-Val-Gly-Lys¹⁵-Lys-Arg-Arg-Pro-Val²⁰-Lys-Val-Tyr-Pro-Asn²⁵-Gly-Ala-Glu-Asp-Glu³⁰-Ser-Ala-Glu-Ala-Phe³⁵-Pro-Leu-Glu-Phe³⁹-Ac (SEQ ID NO: 1) ("hACTH"), wherein N and Ac represent the amino and carboxy termini of the molecule, respectively. ACTH (1-24) is conserved because it is present in many chordaeACTH (1-24) was found, including humans, with the following peptide sequence: N-Ser¹-Tyr-Ser-Met-Glu⁵-His-Phe-Arg-Trp-Gly¹⁰-Lys-Pro-Val-Gly-Lys¹⁵-Lys-Arg-Arg-Pro-Val²⁰Human ACTH (1-24) (and the same 1-24 portion of murine ACTH ("mACTH (1-24)") of-Lys-Val-Tyr-Pro-Ac (SEQ ID NO: 2) where N and Ac represent the amino and carboxy termini of the molecule, respectively.

ACTH analog compounds can include at least amino acid residues 1-19, more preferably amino acid residues 1-24 of hACTH (SEQ ID NO: 1) with one or more amino acid substitutions. Preferred ACTH analog compounds comprise seq id NO: 2. ACTH analog compounds include compounds that include an ACTH sequence with one or more structural modifications that provide one or more of the following preferred ACTH analog biological functions: (1) decreased secretion of corticosteroids by the adrenal membrane in the presence of ACTH analogs as compared to unmodified ACTH, (2) decreased secretion of corticosteroids by the adrenal membrane in the presence of endogenous ACTH, and (3) increased binding affinity for MC-2R with decreased activation of MC-2R receptors as compared to unmodified ACTH.

Particularly preferred ACTH analog compounds have one or more of the following amino acid substitutions to the unmodified human ACTH sequence: (1) one or more amino acid residue substitutions at positions 1-13 that retain the amino acids at positions 6-9, (2) one or more amino acid residue substitutions at these positions that prevent or oppose enzymatic cleavage at positions 15-18, (3) one or more amino acid residue substitutions at positions 15-18 that prevent the occurrence of two adjacent basic amino acids, (4) one or more amino acid residue substitutions or truncations at positions 20-24 that extend the serum half-life of the ACTH analog, and (5) one or more amino acid residue substitutions or preferably truncations at positions 25-39 that provide an extended serum half-life to the ACTH analog compound (as compared to unmodified ACTH or as compared to other ACTH analog compounds).

ACTH analogs preferably comprise a polypeptide described by the following formula (I):

(I)N-(AA^1-13)-(AA¹⁴)-(AA^15-18)-(AA¹⁹)-Ac

wherein N and Ac refer to the amino-and carboxy-terminus of the polypeptide, respectively, (AA)^1-13) Refers to the first series of 13 consecutive amino acids or amino acid analogues, (AA)¹⁴) Represents the amino acid residue attached to the carboxy terminus of the first series, (AA)^15-18) Finger attached to (AA)¹⁴) The second series of 4 consecutive amino acids at the carboxy terminus, (AA)¹⁹) Represents the amino acid residue attached to the carboxy terminus of the second series.

ACTH analogs of formula (I) also preferably comprise a linkage to (AA)¹⁹) A portion of the carboxy terminus of the-Ac moiety is a larger molecule. ACTH analogs may also include additional amino acids attached to the carboxy terminus (Ac) of formula (I). Most preferably, ACTH analogs include a total of 5 additional amino acids attached to the Ac moiety of formula (I), such that there are a total of 24 amino acids in the ACTH analog.

ACTH analogs can comprise an amino acid sequence corresponding to unmodified ACTH of formula (I), preferably including one or more amino acid substitutions. The further amino acid or amino acid analogue is preferably linked to (AA) of the formula (I)¹⁹) The carboxyl terminus of the residue.

(AA) of the formula (I)^1-13) -a sequence of 13 amino acids, partially representing formula (II):

(II)-AA¹-AA²-AA³-AA⁴-AA⁵-AA⁶-AA⁷-AA⁸-AA⁹-AA¹⁰-AA¹¹-

AA¹²-AA¹³-

having an unmodified ACTH amino acid sequence as set forth in the second row of table 1, optionally including one or more amino acid substitutions as provided in the third row of table 1. Unmodified human ACTH (AA)^1-13) -a moiety having the sequence Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val (SEQ ID NO: 3). Preferably, AA of formula (II)⁶-AA⁷-AA⁸-AA⁹The portion has the unmodified amino acid sequence His-Phe-Arg-Trp- (SEQ ID NO: 4) optionally substituted by one or more of its (D) amino acid analogs.

In table 1, the label (D) refers to the (D) enantiomer of the indicated amino acid; nle refers to the amino acid analog norleucine or another amino acid with an alkyl side chain; orn refers to ornithine or another modified amino acid with a similar side chain; dab refers to 2,4 diaminobutyric acid or a similar diamino acid; dpr refers to 2, 3 diaminopropionic acid or another diamino acid; (D) p-iodo-Phe refers to a steric group (steric group), such as Phe modified for iodine; and (D) -I-naph-Ala means I-naphthyl modified (D) -Ala or another sterically modified amino acid. Including unmodified (AA) to unmodified ACTH^1-13) Partial substitution ACTH analog compounds preferably include one of the optional ACTH analog amino acid residues indicated below the residue positions given in table 1. For example, unmodified ACTH is in position AA⁵Has a Glu residue which may be optionally replaced by a (D) -Glu, Cys or Asp residue in the formed ACTH analogs. The amino acid sequence of formula (II) may also include one or more amino acid substitutions corresponding to the MC-2R binding sequence described by Hruby et al in U.S. Pat. No. 4,485,039; 4,457,864, respectively; 4,866,038, respectively; 5,731,408, respectively; 5,714,576, respectively; 5,049,547, respectively; 4,918,055, respectively; 4,649,191 and 5,674,839, which are incorporated herein by reference.

(AA) of the formula (I)¹⁴) The-moiety represents an amino acid residue, which is preferably Gly or (D) Gly, but retains the good biological functions of ACTH analog compounds (e.g., reduced secretion of corticosteroids produced by ACTH analogs, reduced flux, as compared to ACTH)Secretion of corticosteroids by endogenous ACTH and/or MC-2R binding) may also be present (AA)¹⁴) -position substitution.

(AA) of the formula (I)^15-18) -a sequence of 4 amino acids partially representing formula (III):

(III)-AA¹⁵-AA¹⁶-AA¹⁷-AA¹⁸-。

unmodified human ACTH (AA)^15-18) -a portion comprising the sequence Lys-Arg (SEQ id no: 5) it comprises 4 adjacent amino acids with basic side chains. Preferably, ACTH analog compounds include one or more amino acid residue substitutions in formula (III) that prevent or oppose enzymatic cleavage at these sites. Also preferably, ACTH analog compounds include one or more amino acid substitutions in formula (III) that avoid the presence of adjacent amino acids with basic side chains. Preferably, AA¹⁵And AA¹⁶-is independently selected from Lys, Ala, Gly and Val; preferably, AA¹⁷-AA¹⁸Independently selected from Arg, Ala, Gly, and Val.

In one aspect, the nucleic acid sequence of SEQ ID NO: 2 or a residue AA of the formula (III)¹⁶-AA¹⁷-AA¹⁸Such that amino acid residues 16, 17 and 18 of the ACTH analog do not include 2 adjacent amino acid residues selected from Lys and Arg. In other words, in some ACTH analogs, the peptide sequence AA¹⁶-AA¹⁷-AA¹⁸The amino acids Lys and Arg in the moiety cannot occur adjacent to each other (i.e. one arginine adjacent to one lysine, and vice versa) or adjacent to itself (i.e. one arginine adjacent to another arginine, or one lysine adjacent to another lysine). Alternatively, AA¹⁶-AA¹⁷-AA¹⁸One or more amino acids in the moiety may be replaced with any amino acid or amino acid analog other than Lys or Arg to provide one or more of the following preferred ACTH analogs biological functions: (1) reduced adrenal membrane-to-skin in the presence of ACTH analogs compared to unmodified ACTHSecretion of a corticosteroid, (2) reduction of corticosteroid secretion by adrenal membrane in the presence of endogenous ACTH, and (3) increased binding affinity for MC-2R with reduced activation of MC-2R receptors compared to unmodified ACTH. For example, ACTH analog compounds can include an Ala residue in place of an unmodified ACTH residue at one or more positions in formula (III). ACTH analog compounds may include an amino acid sequence that replaces SEQ ID NO: 5 residues Gly or any amino acid with an alkyl side chain (i.e. Ala, Val, Leu, Ile or amino acid analogues containing an alkyl side chain, such as Nle). Also preferably, ACTH analogs can further include a substitution in place of SEQ ID NO: 2 at least one Ala residue and at least one Arg residue at any two of amino acid positions 15, 16, 17 or 18. Optionally, in SEQ ID NO: 2 is an amino acid selected from the group consisting of: lys, Arg, Ala, Gly, Val, Leu, Ile, amino acid analogs comprising alkyl side chains such as Nle, Gln, Asn, Glu, and Asp. Most preferably, ACTH analogs comprise an amino acid sequence according to formula (III) selected from the group consisting of: Lys-Arg-Ala-Ala- (SEQ ID NO: 6), Ala-Lys-Ala-Arg (SEQ ID NO: 7), Lys-Ala-Ala-Arg (SEQ ID NO: 8), Lys-Ala-Arg-Ala- (SEQ ID NO: 9), Gln-Lys-Gln-Arg (SEQ ID NO: 10), and Ala-Ala-Ala (SEQ ID NO: 11). ACTH analog compounds may also include one or more amino acid residue substitutions at positions 15, 16, 17, or 18 with amino acids or amino acid analogs having alkyl side chains, including Gly, Ala, Val, Leu, Ile, or Nle. ACTH analogs are included in AA¹⁵-AA¹⁶-AA¹⁷-AA¹⁸Substitutions at one or more of the positions with other amino acids that retain one or preferably more of the biological functions of the ACTH analog compounds, such as reducing corticosteroid secretion by ACTH analogs compared to unmodified ACTH, reducing corticosteroid secretion by endogenous ACTH and/or MC-2R binding.

(AA) of the formula (I)¹⁹) The moiety represents an amino acid residue which may be Pro, Trp or Ala, but is preferably Trp, Ala or may be (AA)¹⁹) -bitOther amino acids that can be substituted while retaining one or preferably more of the biological functions of the ACTH analog compounds (e.g., reducing corticosteroid secretion by ACTH analogs, reducing corticosteroid secretion by endogenous ACTH and/or MC-2R binding as compared to unmodified ACTH). Preferably, the ACTH analog compound has a structure that can be represented at (AA)¹⁹) -other amino acids than proline substituted in position. More preferably, the ACTH analogs are included in (AA)¹⁹) -the amino acid Trp substituted in position instead of Pro. Unmodified ACTH peptide sequences, such as hACTH and mACTH, comprise (AA)¹⁹) -a proline residue at position. Proline has the following chemical structure:

proline (Pro)

The chemical structure of proline includes a side chain that forms a ring structure. Proline is often present at the end of the alpha helix or in the turn or loop. Unlike other amino acids, which exist in almost the only trans form in a polypeptide, proline may exist in the peptide in the cis configuration. Cis and trans are almost isopotent. Preferably, the nucleic acid sequence comprising SEQ ID NO: ACTH analogs of 6-10 (AA)¹⁹) Is Trp, but also among the ACTH Analogues (AA)¹⁹) -is Pro.

Thus, particularly preferred ACTH analogs are modified hACTH, mACTH or hACTH (1-24) peptide sequences wherein (AA)¹⁴)-(AA^15-18)-(AA¹⁹) -is selected from: -Gly¹⁴-Lys¹⁵-Arg¹⁶-Ala¹⁷-Ala¹⁸-Trp¹⁹-(SEQ ID NO：12)；-Gly¹⁴-Ala¹⁵-Lys¹⁶-Ala¹⁷-Arg¹⁸-Pro¹⁹-(SEQ ID NO：13)；-Gly¹⁴-Lys¹⁵-Ala¹⁶-Ala¹⁷-Arg¹⁸-Pro¹⁹-(SEQ ID NO：14)；-Gly¹⁴-Lys¹⁵-Ala¹⁶-Arg¹⁷-Ala¹⁸-Pro¹⁹-(SEQ ID NO：15)；-Gly₁₄-Gln¹⁵-Lys¹⁶-Gln¹⁷-Arg¹⁸-Pro¹⁹- (SEQ ID NO: 16) and-Gly¹⁴-Lys¹⁵-Arg¹⁶-Ala¹⁷-Ala¹⁸-Pro¹⁹-(SEQ ID NO：17)。

ACTH analogs comprise a polypeptide of formula (IVa) or formula (Ivb):

(IVa)N-(AA^1-13)-(AA¹⁴)-(AA^15-18)-(AA¹⁹)-(AA^20-24)-

(IVb)N-(AA^1-13)-(AA¹⁴)-(AA^15-18)-(AA¹⁹)-(AA^20-24)-Ac

wherein N-refers to the N-terminus of the polypeptide and Ac refers to the carboxy-terminus of the polypeptide. ACTH analogs of formula (VIa) or (VIb) include the N- (AA) described for formula (I) above^1-13)-(AA¹⁴)-(AA^15-18)-(AA¹⁹) Furthermore, (AA) linked to the carboxy terminus of the amino acid sequence of the formula (I)^20-24) -a moiety. The (AA20-24) -moiety of formulae (VIa) and (VIb) represents a5 amino acid sequence of formula (V):

(V)-AA²⁰-AA²¹AA²²AA²³-AA²⁴-。

unmodified human ACTH (AA)^20-24) -a moiety comprising the sequence Val-Lys-Val-Tyr-Pro (seq id NO: 18). Preferably, ACTH analog compounds include one or more amino acid residue substitutions in formula (V) that extend the serum half-life of the ACTH analog. For example, ACTH analogs can include the sequence corresponding to formula (V) -Ala-Ala-Ala-Ala-Ala- (SEQ ID NO: 19).

Particularly preferred ACTH analogues according to formula (IVa) comprise the sequence hACTH (1-24) comprising the sequence defined by SEQ ID NO: 6 substituted formula (III) -AA¹⁵-AA¹⁶-AA¹⁷-AA¹⁸-a moiety. A special featureFurther preferred compounds of formula (IVa) are ACTH analogs (KKRRP15-19KRAAW) mACTH (1-24) having the sequence N-Ser¹-Tyr-Ser-Met-Glu⁵-His-Phe-Arg-Trp-Gly¹⁰-Lys-Pro-Val-Gly-Lys¹⁵-Arg-Ala-Ala-Trp-Val²⁰-Lys-Val-Tyr-Pro-Ac (SEQ ID NO: 20), also known as "AT 814". Other particularly preferred ACTH analog compounds consist essentially of SEQ ID NO: 2. For example, Costa, JL et al, "microbiological analysis of organic consistent ACTH residues," Gen Comp Endocrinol. Mar; 136(1): 12-6(2004), which is incorporated herein by reference in its entirety.

In a third aspect of the first embodiment, ACTH analogs comprise a polypeptide described by formula (VIa) or formula (VIb):

(VIa)N-(AA^1-13)-(AA¹⁴)-(AA^15-18)-(AA¹⁹)-(AA^20-24)-(AA^25-39)-

(VIb)N-(AA^1-13)-(AA¹⁴)-(AA^15-18)-(AA¹⁹)-(AA^20-24)-(AA^25-39) -Ac wherein N-refers to the amino terminus of the polypeptide and Ac refers to the carboxy terminus of the polypeptide. ACTH analogs of formula (IVa) or (IVb) include N- (AA) described above for formula (IVa)^1-13)-(AA¹⁴)-(AA^11-18)-(AA¹⁹)-(AA^20-24) And further comprising (AA) attached to the carboxy terminus of the amino acid sequence of formula (IVa)^25-39) -a moiety. Formula (VIa) may optionally include attachment to AA³⁹Additional chemical structure of residue, wherein AA³⁹The residue forms the carboxy terminus of the structure of formula (VIb). (AA) of formulae (VIa) and (VIb)^25-39) -a sequence of 15 amino acids partially representing formula (VII):

(VII)-AA²⁵-AA²⁶-AA²⁷-AA²⁸-AA²⁹-AA³⁰-AA³¹AA³²-AA³³-AA³⁴-

AA³⁵-AA³⁶-AA³⁷-AA³⁸-AA³⁹。

unmodified human ACTH (AA)^25-39) -the moiety comprising the sequence Asn-Gly-Ala-Glu-Asp-Glu-Ser-Ala-Glu-Ala-Phe-Pro-Leu-Glu-Phe (SEQ ID NO: 21). Preferably, the ACTH analog compound includes one or more amino acid residue substitutions in formula (VII) or one or more amino acid residue truncations at the carboxy terminus of formula (VII), so long as the ACTH analog compound has the desired sustained release characteristics. For example, ACTH analogs can comprise a sequence corresponding to formula (VII) having the sequence of SEQ ID NO: 10 and may optionally be via AA³⁰Lys replacement and/or AA of³⁶An Arg substitution modification of (1).

Substitutional variants are those in which at least one residue in the amino acid sequence is removed and a different residue is inserted in its place. If fine tuning of the protein properties is desired, such substitutions can be made according to Table 2 below. Table 2 lists amino acids that can be substituted for the original amino acids in the protein and are considered conservative substitutions in the art. ACTH analog compounds include compounds with one or more conservative substitutions that retain one or more of the following preferred ACTH analog biological functions: (1) decreased secretion of corticosteroids by the adrenal membrane in the presence of ACTH analogs as compared to unmodified ACTH, (2) decreased secretion of corticosteroids by the adrenal membrane in the presence of endogenous ACTH, and (3) increased binding affinity for MC-2R with decreased activation of MC-2R receptors as compared to unmodified ACTH.

TABLE 2

Substitution changes in functional or immunological properties are made by selecting substitutions that are less conservative than those in table 2, i.e., residues are selected that differ more significantly in maintaining the following properties: (a) a polypeptide backbone structure of the replacement region, e.g., in a folded or helical conformation; (b) charge or hydrophobicity of the molecule at the target site; or (c) side chain volume. It is generally expected that the substitutions that produce the greatest changes in protein properties will be those in which: (a) replacement of a hydrophobic residue such as leucine, isoleucine, phenylalanine, valine, or alanine with a hydrophilic residue such as serine or threonine (or replacement with a hydrophobic residue such as leucine, isoleucine, phenylalanine, valine, or alanine); (b) cysteine or proline for any other residue (or by any other residue); (c) substitution of (or substitution of) a negatively charged residue such as glutamic acid or aspartic acid with a residue having a positively charged side chain such as lysine, arginine or histidine; or (d) a residue with a bulky side chain such as phenylalanine in place of (or in place of) a residue without a side chain such as glycine.

Preferred ACTH analog biological functions of ACTH analog compounds can be measured by any suitable method, but are preferably performed by one or more of the assays described below.

ACTH analog compounds with reduced ACTH function

In a second embodiment, the ACTH analogs are modified ACTH peptides that reduce corticosteroid secretion by adrenal membranes in the presence of the ACTH analog compared to unmodified ACTH. The structure of ACTH analogs is preferably selected according to one or more of the structural formulae provided above. ACTH analog compounds can reduce ACTH-mediated secretion of blood corticosterone, as indicated by a reduction in blood corticosteroid levels in a subject following administration of the ACTH analog. ACTH analogs preferably reduce serum corticosterone levels by at least 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% compared to comparable administration of unmodified ACTH peptide, as determined by an in vivo serum corticosteroid induction assay.

In vivo serum corticosteroid induction assays are used to measure the level of corticosteroid or corticosterone in the blood stream of a subject. More specifically, the in vivo serum corticosteroid induction assay may comprise the following steps: first, a mouse subject is administered an agent (agent) that inhibits endogenous ACTH production, e.g., intraperitoneal injection of dexamethasone; second, after waiting an appropriate period of time (e.g., 1.5-2.0 hours) to inhibit endogenous ACTH production, the test compound is administered to the mouse by an appropriate method (e.g., intraperitoneal injection or subcutaneous injection); third, after waiting for the injected test compound to act for a suitable period of time (e.g., 1 hour), a blood sample is obtained from the subject and passed through a suitable method, e.g., using¹²⁵Competitive radioimmunoassay of the RIA kit (ICN, Costa Mesa, CA) determines serum corticosterone levels. Example 1 describes in detail the in vivo serum corticosteroid induction assay in mice.

Corticosteroid or corticosterone secretion induced in vivo by ACTH analog compounds is compared to the level of corticosteroid secretion produced by unmodified ACTH compounds by measuring serum corticosteroids after ACTH or ACTH analogs are administered as test compounds. Figure 1 shows the results of a murine in vivo corticosteroid adrenal induction assay performed using mACTH (1-24) and various ACTH analog test compounds according to example 1. ACTH analog compositions with reduced function were identified by measuring corticosterone levels in blood samples collected 1 hour after administration of mACTH (1-24) or ACTH analog compounds to dexamethasone-inhibited mice using standard Radioimmunoassay (RIA). In figure 1, potency of various ACTH analog peptides is expressed as a percentage of corticosterone induction, where the potency of murine ACTH is considered to be 100%. Graph 10 shows the percent corticosterone induction 12 for ACTH and 9 ACTH analog compound samples 14. Percent corticosterone induction 12 of ACTH analog compounds in samples 2-10 is shown as a percent 20 of the serum corticosterone concentration of unmodified mACTH (1-24) as measured by the in vivo serum corticosteroid adrenal induction assay. For samples 2-10 in FIG. 1, the structural modifications and the percent reductions measured are shown in Table 3 below.

TABLE 3

Endogenous ACTH-induced corticosterone-induced reduction

In a third embodiment, ACTH analogs are modified ACTH peptides that reduce corticosteroid secretion by adrenal membrane in the presence of endogenous ACTH. ACTH analog compounds can reduce serum corticosteroid levels by 10-100% when administered prior to or concurrently with ACTH. Preferably, administration of the ACTH analog compound reduces serum corticosteroid levels by about 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% as compared to ACTH alone, as determined by an in vivo serum corticosteroid inhibition assay, such as the assay described in example 2. Exemplary ACTH analog compounds that inhibit endogenous ACTH-induced adrenal hormone production were identified by performing a series of in vivo serum corticosteroid inhibition assays. ACTH analogs can be administered alone prior to ACTH, or concurrently with ACTH.

The level of corticosteroid or corticosterone in the blood stream of a subject following administration of a compound having a first corticosteroid-producing biological activity and a test compound was measured using an in vivo serum corticosteroid inhibition assay. The compound having corticosteroid-producing biological activity can be any compound known to induce a detectable increase in corticosteroid production. The test compound may be administered in combination with the corticosteroid-producing compound, and the test compound may be administered prior to administration of the corticosteroid-producing compound and/or after administration of the corticosteroid-producing compound. The corticosteroid-producing compound is preferably ACTH, but may also be an ACTH analog having known corticosteroid-producing biological activity. By performing a series of in vivo serum corticosteroid inhibition assays using ACTH analog test compounds, ACTH analogs that inhibit corticosteroid production relative to the corticosteroid-producing compound used can be identified. Preferably, ACTH analogs identified in this manner inhibit corticosteroid production in the presence of endogenous, unmodified ACTH.

The in vivo serum corticosteroid inhibition assay may comprise sequential administration of a test compound and a corticosteroid-producing compound by: first, a mouse subject is administered an agent that inhibits endogenous ACTH production, e.g., intraperitoneal injection of dexamethasone; second, after waiting an appropriate period of time (e.g., 1.5-2.0 hours) to inhibit endogenous ACTH production, the test compound is administered to the mouse by an appropriate method (e.g., intraperitoneal injection); third, after waiting for the test compound to act for an appropriate period of time (e.g., 1 hour), the compound having corticosteroid-producing activity is administered to the mouse by an appropriate method (e.g., intraperitoneal injection); and fourth, after waiting for the appropriate period of time (e.g., 1 hour) for the biological activity of the two compounds administered to occur, a blood sample is obtained from the subject and treated by an appropriate method, e.g., using¹²⁵Competitive radioimmunoassay of the RIA kit (ICN, Costa Mesa, CA) determines serum corticosterone levels. Optionally, steps 2 and 3 may be reversed (first administering the corticosteroid-producing compound, then administering the test compound). The in vivo serum corticosteroid inhibition assay may further comprise co-administering the test compound and the corticosteroid-producing compound by: first, a mouse subject is administered an agent that inhibits endogenous ACTH production, e.g., intraperitoneal injection of dexamethasone; second, after waiting an appropriate period of time (e.g., 1.5-2.0 hours) to inhibit endogenous ACTH production, the corticosteroid-producing compound and the test compound are co-administered to the mice by an appropriate method (e.g., intraperitoneal injection); and third, the biology of the two compounds waiting to be administeredAfter an appropriate period of time (e.g. 1 hour) for the activity to occur, a blood sample is obtained from the subject and passed through an appropriate method, e.g. using¹²⁵Competitive radioimmunoassay of the RIA kit (ICN, Costa Mesa, CA) determines serum corticosterone levels. Likewise, in some embodiments, rather than administering an agent that inhibits ACTH production, the first step in any in vivo serum corticosteroid inhibition assay may be replaced by measuring the initial endogenous corticosteroid or corticosteroid level in the subject's blood in the manner of step 4. Example 2 describes in detail the in vivo serum corticosteroid inhibition assay in mice.

A series of individual in vivo serum corticosteroid inhibition assays were performed to identify ACTH analogs that inhibit ACTH adrenal hormone production in vivo. Serum corticosterone levels in dexamethasone-inhibited mice were determined as described in example 2 after administration of (1) ACTH (corticosteroid-producing compound), (2) ACTH analog (test compound), followed by ACTH, (3) ACTH and ACTH analog combination, and (4) ACTH analog alone. Table 4 shows the measured serum corticosteroid levels of the mice as a function of the time of administration.

TABLE 4 Combined administration of ACTH and ACTH analogs

With respect to table 4, dexamethasone was first administered to 5 independent mice in an in vivo serum corticosteroid assay at time-0 min as described in example 2. In the "vehicle" sample, the liquid vehicle alone was administered 120 minutes after dexamethasone injection, with the result that no corticosterone was detected in the blood sample after 1 hour. In the "ACTH" samples, ACTH was administered 120 minutes after dexamethasone injection, resulting in corticosterone levels of 500 + -76 ng/mL detected in blood samples after 1 hour. In the "AT 90-ACTH 120" sample, ACTH analog (KKRRP15-19KRAAW) mACTH (1-24) was administered 1.5 hours after dexamethasone injection, followed by ACTH (1-24) after 30 minutes, resulting in a detected serum corticosterone level of 175 + -27 ng/mL after 1 hour. In the "(AT + ACTH) 120" samples, ACTH analogs (KKRRP15-19KRAAW) mACTH (1-24) ("AT 814") and mACTH (1-24) were administered simultaneously 120 minutes after dexamethasone injection, resulting in a serum corticosterone level of 51 + -8 ng/mL after 1 hour. In the "AT" samples, ACTH analog (KKRRP15-19KRAAW) mACTH (1-24) was administered 120 minutes after dexamethasone injection, resulting in negligible serum corticosterone levels of 7. + -.7 ng/mL.

Figure 2 shows the results from table 2 expressed as a percentage of the corticosterone level determined at 180 minutes of dexamethasone injection as described in example 2. Graph 100 shows the percent corticosterone induction for ACTH and 4 samples 114 described in table 2, where corticosterone levels 120 measured for administration of ACTH were normalized to 100%. Sample "AT 90-ACTH 120" 140 showed that administration of ACTH analogs 30 minutes prior to ACTH administration resulted in approximately 65% reduction in serum corticosterone levels in blood samples AT 1 hour post ACTH administration. Sample "(AT + ACTH) 120" 160 shows that concurrent administration of ACTH analog and ACTH results in a decrease in serum corticosterone levels in the blood sample of approximately 90% after 1 hour. As seen in "AT" sample 180 and sample 10 in table 1, administration of ACTH analog alone resulted in a decrease in serum corticosterone levels in the blood sample by approximately 99% -100% after 1 hour. The "carrier" sample is not shown in figure 2.

Adrenal ACTH receptor binding assays

In a fourth embodiment, the ACTH analogs are modified ACTH peptides that bind to an adrenal ACTH receptor, e.g., the MC-2R receptor, preferably with increased binding affinity to MC-2R but with decreased activation of the MC-2R receptor, as compared to the unmodified ACTH.

Preferred ACTH analog compounds bind to adrenal membrane with greater affinity than endogenous unmodified ACTH and are capable of replacing unmodified ACTH in an in vitro serum-free adrenal competition binding assay (example 3). ACTH analog compounds preferably have at least 20% displacement of ACTH bound to adrenal membrane preparations as determined by an in vitro serum-free adrenal competition binding assay. More preferably, ACTH analog compounds have a2, 3, 4,5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 fold greater affinity for explanted adrenal membrane than unmodified ACTH.

ACTH analog compounds that bind to adrenal membrane with greater affinity than endogenous unmodified ACTH and can replace unmodified ACTH can be identified using an in vitro serum-free adrenal competition binding assay (example 3). Fig. 3 shows the use of an illustrative polypeptide having SEQ ID NO: 20 ACTH analog AT814 ("AT 814") results from an in vitro serum-free adrenal competition binding assay performed by the method described in example 3. Graph 200 shows counts per minute 212(CPM) measured by a gamma counter counting adrenal membrane preparations incubated with radioactive ACTH. A higher level of CPM indicates a greater amount of radiolabeled compound present. Bar 210 shows the background value measured in the absence of any adrenal membrane. Bar 220 shows the level of radiolabeled unmodified mACTH (1-24) detected 2 hours after mixing of explanted adrenal membrane with radiolabeled mACTH (1-24) (no competitive binding). Bars 230, 240 and 250 show the results of a competitive binding assay, in which a non-radiolabeled ("non-labeled") mACTH (1-24) displaces labeled mACTH (1-24) from the explanted adrenal membrane by competitive binding to a receptor such as MC-2R on the adrenal membrane (measured in bar 220). Specifically, bar 230 shows the decrease in radiolabeled mACTH (1-24) levels detected after mixing radiolabeled explanted adrenal membranes with 10nM "unlabeled" mACTH (1-24); bar 240 shows a further reduction in the level of radiolabeled mACTH (1-24) bound to radiolabeled explant adrenal membrane after addition of 100nM "unlabeled" mACTH (1-24); and bar 250 shows that the level of radiolabeled mACTH (1-24) attached to radiolabeled explanted adrenal membranes was further reduced after mixing the explanted adrenal membrane preparation with 1000nM of "unlabeled" mACTH (1-24). As the concentration of the "unlabeled" mACTH (1-24) increased, the detected radiolabeled mACTH (1-24) decreased, indicating that the "unlabeled" mACTH (1-24) displaced the radiolabeled mACTH (1-24) from the MC-2R receptor.

Using a peptide consisting of SEQ ID NO: the in vitro serum-free adrenal competition binding assay was repeated with the "unlabeled" (non-radiolabeled) AT814ACTH analog compound described in 20 in place of mACTH (1-24). Columns 235, 245 and 255 show the results of a competitive binding assay, where the non-radiolabeled AT814 displaces the radiolabeled mACTH (1-24) from explanted adrenal membranes (measured in column 220) by competitive binding of "non-labeled" AT814 AT concentrations of 10nM (column 235), 100nM (column 245) and 1000nM (column 255). As the concentration of "non-labeled" AT814 increased, the level of detected radiolabeled AT814 decreased, indicating that "non-labeled" AT814 displaced the radiolabeled mACTH (1-24). Specifically, the results in graph 200 indicate that AT814 binds to adrenal membrane with an affinity that is about 3 to about 4 times greater than the affinity of radiolabeled mACTH (1-24) for adrenal membrane binding. An increase in the affinity of AT814 for binding to adrenal membrane predicts an increase in affinity for binding to MC-2R receptors.

In vitro testing of ACTH analog compounds

In a fifth embodiment, ACTH analog compounds can reduce the in vitro induction of corticosterone by unmodified ACTH in explant tissue. Preferably, the serum-free medium reduces the levels of the steroid by 10-100% when the explanted adrenal membrane is mixed with the ACTH analog compound. Preferably, ACTH analog compounds reduce serum corticosteroid levels by about 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% as determined by an in vitro serum-free adrenal corticosteroid inhibition assay as compared to administration of unmodified ACTH alone (example 4). Exemplary ACTH analog compounds that inhibit adrenal hormone production induced by explanted adrenal membrane contact with unmodified ACTH were identified by a series of in vitro serum-free adrenocorticosterone inhibition assays, indicating that ACTH analog compounds have a direct effect on adrenocorticosterone production.

A series of in vitro serum-free adrenocortical steroid inhibition assays were performed to identify ACTH analogs that inhibit ACTH-induced adrenohormonal production in vitro. Cortisone concentrations were measured in serum-free media containing explanted adrenal membrane and unmodified ACTH, AT814ACTH analog (SEQ ID NO: 20), or ACTH in combination with AT814 (100 ng/mL each) following the procedure in example 4. Table 3 shows the corticosteroid levels measured in serum-free medium in a series of in vitro serum-free adrenocorticotropin inhibition assays performed using explanted mouse adrenal membranes. Corticosterone levels were determined 2.0 hours after the adrenal membrane was placed in serum-free M199 medium, and each corticosterone level in table 5 was the average of 5 samples.

TABLE 5 Combined administration of ACTH and ACTH analogs

With respect to table 5, half of the explanted mouse adrenal glands were placed in M199 serum-free medium (Invitrogen) for 30 minutes as provided in example 4. In the "M199 media" samples, after the initial 30 minutes, adrenal membranes were soaked in M199 serum-free media for a total of 2.0 hours (total of 2.5 hours), and then corticosterone levels in the media were measured using standard RIA radiometric techniques. In the "ACTH" samples, unmodified ACTH was added to M199 medium, resulting in corticosterone levels detected in the medium after 2 hours of approximately 600 mg/mL. In the "AT-814" sample, ACTH analog (KKRRP15-19KRAAW) mACTH (1-24) (SEQ ID NO: 20) was added to M199 medium, resulting in a corticosterone level detected in the medium after 2 hours of approximately 290 mg/mL. In the "ACTH + AT-814" sample, 100ng/mLAT814ACTH analog (SEQ ID NO: 20) was added to M199 medium along with 100ng/mL unmodified ACTH, resulting in a corticosterone level of about 396mg/mL detected in the medium after 2 hours. Figure 4 is a graph 300 showing the percent corticosterone induction 302 from table 3 expressed as a percentage of the corticosterone level in M199 medium relative to the amount 310 measured for ACTH as determined for the "ACTH" sample 310, the "AT-814" sample 320, and the "ACTH + AT-814" sample 330 as described above and in example 4. The "in M199 medium" sample is not shown in FIG. 2. With respect to fig. 4, the addition of AT814 to the adrenal membrane in M199 serum-free medium induced a negligible amount of corticosterone, with a combined addition of equal amounts of AT814ACTH analog and unmodified ACTH resulting in a reduction of the induced corticosterone by approximately 65% compared to the addition of unmodified ACTH alone.

In vivo testing of ACTH analog compounds

For certain applications, it is preferred to use ACTH analog compounds with extended serum half-lives. ACTH analog compounds can optionally include one or more amino acid substitutions or truncations that increase the serum half-life of the ACTH analog compared to unmodified ACTH or another ACTH analog. For example, ACTH analog compounds can include one or more amino acid substitutions in amino acid positions 25-39 of the unmodified ACTH sequence such that the serum half-life of the compound is extended compared to the ACTH sequence. Preferred ACTH analog compounds may have a longer serum half-life than unmodified ACTH. In blood, ACTH has a half-life of less than about 20 minutes. Thus, particularly preferred ACTH analog compounds have a serum half-life of greater than about 20, 30, 40, or 50 minutes or more.

In a sixth embodiment, extended half-life ACTH analogs are provided. ACTH analogs having halochenne half-lives are identified as analogs in which a first activity, as measured by serum corticosteroid concentration measured in vivo, is greater than a second activity, as measured by serum-free corticosteroid concentration measured in vitro, wherein the in vivo activity is measured by the serum adrenal corticosteroid inhibition assay of example 1 and the in vitro activity is measured by the in vitro serum-free adrenal corticosteroid inhibition assay of example 4.

In some cases, amino acid substitutions at other positions can also extend the serum half-life of the ACTH analog, and thus certain embodiments extend the serum half-life of the ACTH analog by making one or more amino acid modifications at positions 20-24 of the ACTH molecule, including hACTH or mACTH. Figure 5 compares the in vivo activity (grey bars) and in vitro activity (white bars) of 7 ACTH analog compounds 410, 420, 430, 440, 450, 460, and 470. For all 7 ACTH analog compounds, in vivo activity was determined using the method of example 1, and in vitro activity was determined using the method of example 4. The results are presented as a plot of the measured percentage "% corticosterone induction" relative to the mACTH (1-24) sequence (SEQ ID NO: 2). As seen in sample 460 of FIG. 5, the designated "Ala 19-24" refers to the ACTH analog (AAAAA20-24VKVYP) ACTH (1-24) which includes Ala residue substitutions at each of amino acids 19-24 of mACTH. Sample 460 shows an increase in serum half-life 462, relative to the in vitro activity 464 of this compound, as measured in vivo, and is approximately 50% greater than the in vitro activity of unmodified mACTH. In addition, the in vivo activity of (AAAAA20-24VKVYP) ACTH (1-24) ACTH analog 460 was higher than that of mACTH and other ACTH analogs 410, 420, 430, 440, 450, and 470. These results indicate that amino acid residues at positions 20-24 may play a role in determining the serum half-life of ACTH analog compounds. Particularly preferred ACTH analog compounds have one or more amino acid substitutions at positions 20-24 of ACTH to increase (relative to ACTH) serum half-life.

It is also contemplated that ACTH analog compounds may include amino acid substitutions that increase serum half-life without increasing at all or more preferably decreasing the induction of corticosterone by the ACTH analog as measured by in vivo (example 1) or in vitro (example 4) assays. For example, substitutions in positions 15-19 in ACTH analogs 410, 420, 430, 440, 450, and 470 reduced the corticosterone-inducing activity of the ACTH analogs in vivo and in vitro as compared to unmodified mACTH (1-24). Specifically, ACTH analog 410 is a (K15A, R17A) mACTH (1-24) ACTH analog, indicating that its in vitro activity 414 is greater than its in vivo activity 412; ACTH analog 420 is a (K16A, R17A) mACTH (1-24) ACTH analog, indicating that its in vitro activity 424 is greater than its in vivo activity 422; ACTH analog 430 is a (K16A, R18A) mACTH (1-24) ACTH analog, indicating that its in vitro activity 434 is greater than its in vivo activity 432; ACTH analog 440 is a (K15Q, R17Q) mACTH (1-24) ACTH analog, indicating that its in vitro activity 444 is greater than its in vivo activity 442; and ACTH analog 470 is a (P19W, K21A) mACTH (1-24) ACTH analog, indicating that its in vitro activity 474 is greater than its in vivo activity.

Screening method

In a seventh embodiment, methods are also provided for screening ACTH analogs for potential use in blocking excess ACTH while maintaining adrenal health. A variety of ACTH analogs can be prepared and administered to patients to evaluate induction of cortisone in vivo.

The molecular recognition specificity of certain compounds (i.e., antigen is recognized by antibody) enables the formation of suitable complexes, which can serve as the basis for analytical immunoassays in liquids and immunosensors on solid phase interfaces. These assays may be ligand binding assays based on observing the product of a ligand binding reaction between a target analyte (i.e., a compound that binds the MC2R receptor) and a highly specific binding reagent.

In some cases, for a particular sensitive screening assay, a selectable analyte-binding compound, such as an aptamer, is used in a ligand-binding assay. Aptamers are single-stranded DNA or RNA oligonucleotide sequences capable of recognizing a variety of target molecules with high affinity and specificity. In various diagnostic formats, these ligand-binding oligonucleotides mimic the properties of antibodies. They fold into a unique overall shape to form complex binding grooves for the target structure. Aptamers are identified by an in vitro selection process called exponential enrichment ligand systemic evolution (SELEX). Aptamers are superior to antibodies in terms of sensitive surface (sensing surface) storage. Furthermore, while the affinity constants are consistently lower than those of antibodies and the stability of these compounds remains questionable, they are particularly useful for screening applications in complex biological metrics (i.e., for screening ACTH analogs having alternative amino acid residues at ACTH- (15-19) and/or ACTH- (21) positions and that are particularly active in binding MC 2R) because of the existence of methods that can be synthesized in any number of highly repetitive manner.

Alternatively, molecular imprinting techniques may be used to screen compounds that affect MC2R activity. This is a technique based on the preparation of polymeric adsorbents, which are selected in advance for a specific substance or structural analog. Functional and cross-linking monomers of plastic materials such as methacrylic and styrene can interact with template ligands (templating ligands) to produce low energy interactions. Then, polymerization was induced. In this process, the molecule of interest is entrapped into the polymer by either a non-covalent self-assembly process or a reversible covalent process. After termination of the polymerization reaction, the template molecule is washed off. The resulting imprint of the template is retained in the rigid polymer and retains the spatial (size, shape) and chemical (specific arrangement of complementary functionalities) memory of the template. Molecularly Imprinted Polymers (MIPs) can bind templates (═ analytes) with a similar specificity to antigen-antibody interactions.

In addition to being used primarily for solid phase extraction and chromatography, molecularly imprinted polymers can also be used as non-biological alternatives to antibodies in competitive binding assays. During the growth of a typical biological cell, carbon-containing nutrients such as glucose are taken up and acid metabolites such as lactic acid are released. These changes in metabolic rate are recorded as changes in the acidification rate of the medium surrounding the cells in a microphysiological recorder (i.e., random-Susman, K.M. et al, "Effects of exogenous expression on metabolic rate of primary pathological amplification cultures: application of silicon microphysiological to neurobiology," J neuros., 12 (3): 773-780 (1992); Baxter, GT. et al, "PKC epsilon is involved in genomic-macroscopic-stimulating factor signal: expression of expression from microbial and antisense oligonucleotide experiments; Biochemical, 31: 19050. biological analyzer et al," B. Biochemical, 3: 3. detection of acidification in culture medium: acidification of acidification rate of medium surrounding the cells; 1093. detection of acidification of culture medium; 1093. B. detection of culture medium; 10954. Cd in D₁-and D₂-transformed fiberstones by silicon microphysiometry, "j.receptor.res., 13 (1-4): 559-571 (1993); and McConnell, h.m. et al, "The biosensor microphysiometer: biological applications of silicon technology, "Science, 257: 1906-1912(1992)). The microphysiological recorder may be used to detect molecules affecting cells. Such molecules include neurotransmitters, growth factors, cytokines, receptors, and the like. Thus, the microphysiometer method can provide valuable information about ACTH analogs that affect MC2R receptor activity.

Synthetic combinatorial libraries can be the source of a wide variety of structures for large-scale biochemical Screening (i.e., Sastry, L. et al, "Screening combinatorial antibody libraries for catalytic acyl transfer reactions," Ciba Found Symp., 159: 145-155 (1991); Persson, MAA. et al, "Generation of differential high-affinity monoclonal antibodies by recombinant binding," Proc. Natl. Acad. Sci. USA, 33: 2432-2436 (1991); and Houghten, R.A., "Generation and use of synthetic peptide libraries for basic research and delivery system," 354-86: 1991). Such libraries are produced by combining liquid and solid phase chemistry and cleaved from the solid support for screening.

Separation techniques such as liquid chromatography, gas chromatography and capillary electrophoresis coupled with mass spectrometry or tandem mass spectrometry have established analytical systems that can be used for structural assessment (i.e., Hsieh, S. et al, "Separation and identification of peptides in single nerves by micro-colloidal n-liquid chromatography-Matrix-assisted laser desorption/ionization-of-flight mass spectrometry and position analysis," analytical chemistry, 70 (9): 1847. 1852 (1998); Tretypalova, N.Y. et al, "Quantitative analysis of 1, 3-branched-DNA analysis in videos and in vision using colloidal chromatography, Mass." concentration J. 126. reaction, Mass. J. et al, "concentration J. 11. J. batch analysis, J. 11. and J. 11. batch analysis, Mass. 11. 3. biological chromatography. 11. 7. batch analysis, Mass. 11. 7. 3. biological chromatography. 3. mass spectrometry, Mass. 3. biological analysis. J. 11. 7. 11. 3. batch. 1. 3. mass. 3. sample J. analysis. 3. sample J. analysis. 7. 3. batch. 3. analysis. 3. batch. 3. analysis. batch. 3. batch. 1. analysis. 3. batch. 3. analysis. batch. 3. analysis. batch. Mass spectrometry is particularly useful in providing molecular weight information for compounds/molecules. With precise and controlled fragmentation of macromolecules, it is also possible to obtain information about the sequence.

Screening ACTH analog compounds to identify compounds that are homologous to SEQ ID NO: 2 or SEQ ID NO: 1, a method of inducing adrenal membrane secretion of a corticosteroid-less ACTH analog compound than does adrenal membrane comprises the step of contacting the adrenal membrane with the ACTH analog. Methods of screening ACTH analog compounds to identify compounds that reduce ACTH-induced corticosteroid secretion include one or more of the following steps:

a. providing a first adrenal membrane and a second adrenal membrane;

b. contacting a first adrenal membrane with a first composition comprising an unmodified peptide comprising an unmodified ACTH peptide, and then determining a first concentration of corticosteroid secreted by the first adrenal membrane after contact with the unmodified ACTH peptide;

c. contacting a second adrenal membrane with a second composition comprising an ACTH analog, and then determining a second concentration of corticosteroid secreted by the second adrenal membrane after contact with the ACTH analog;

d. comparing the first concentration of secreted corticosteroid to the second concentration of secreted corticosteroid; and

e. determining whether the second compound induces less corticosteroid secretion than the first compound.

The unmodified ACTH peptide is preferably SEQ ID NO: 2 or SEQ ID NO: 1.

The first and second adrenal membranes may be located in the subject and the concentration of the corticosteroid is a measured concentration in the blood of the subject (in vivo assay). Alternatively, the first and second adrenal membranes are explanted from the subject and the corticosteroid concentration is the concentration in serum-free medium determined (in vitro assay). A screening assay comprising an in vivo inhibition assay step or an in vitro competitive binding assay step may further comprise the following step, preferably in place of step (c) or after step (c) but before step (d):

a. contacting a second adrenal membrane with a first composition comprising the ACTH peptide and a second composition comprising the ACTH analog simultaneously, and measuring secretion from the second adrenal membrane

A second concentration of corticosteroid.

The screening assay can include the step of performing one or more additional assays including an assay expressed as a serum corticosteroid induction assay, a serum corticosteroid inhibition assay, an adrenal binding assay, or a serum-free adrenal inhibition assay. The steps may be performed in any order, unless otherwise indicated.

ACTH analog compositions and administration

ACTH analogs can be incorporated into pharmaceutical compositions for treating a variety of ACTH-related disorders, including those associated with ACTH overexpression, in patients.

The phrases "pharmaceutically acceptable" or "pharmacologically acceptable" are synonymous and mean that the ligand, material, composition, and/or dosage form is suitable for use in contact with the tissues of humans and animals without additional toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio, within the scope of sound medical judgment.

"therapeutically effective amount" with respect to a compound used in therapy, such as an ACTH analog peptide, refers to the amount of polypeptide or peptide in a formulation that, when administered as part of a dosage regimen of interest (to a chordate, such as a mammal or fish), alleviates symptoms, improves a condition, or slows the onset of a disease, e.g., at a reasonable benefit/risk ratio applicable to any medical treatment, according to clinically acceptable standards for the condition or disease to be treated or for cosmetic purposes.

As used herein, the term "treatment" or any derivative thereof (i.e., treatment) relates to and includes: 1) preventing the development of high levels of an ACTH-associated disease or disorder in a patient who is predisposed to the disease or disorder but is not diagnosed with such a disease or disorder; 2) inhibiting high levels of ACTH-associated disease or disorder, e.g., arresting its development; or 3) alleviating high levels of ACTH-associated diseases or disorders, e.g., resulting in disease or disorder regression.

"patient" or "subject" are synonymous herein and are used to refer to an organism, preferably a chordate such as a mammal, fish or bird, for which treatment is provided according to the invention. Mammals that benefit from the disclosed ACTH analogs and methods of treatment include, but are not limited to, apes, chimpanzees, orangutans, humans, monkeys; and domesticated animals (e.g., pets), such as dogs, cats, mice, rats, guinea pigs, and hamsters. Fish includes salmon and other species used in aquaculture.

The term "ACTH-related disorder" as used herein refers to a disease, disorder, condition or disorder, including certain melanocortin receptor-related disorders, that is treatable by or responsive to modulation of ACTH-mediated corticosteroids or corticosteroid levels, modulation of ACTH binding to adrenal membranes, or modulation of ACTH-receptors such as MC-2R.

The term "melanocortin receptor-associated disorder" as used herein refers to a disease, disorder or condition that can be treated by modulating receptor binding to ACTH in a subject and/or by decreasing corticosteroid levels in a subject. ACTH-related disorders may include "MC-2R-related disorders" or diseases, disorders, or diseases treatable by modulation of MC-2R receptors. Preferably, MC-2R-related disorders can be treated by binding of the MC-2R receptor to ACTH analogs (resulting in reduced levels of secreted corticosteroid compared to levels secreted by endogenous ACTH).

The present invention contemplates all stereoisomers of the compounds identified using the methods of the invention, including enantiomeric and diastereomeric forms. The individual stereoisomers of the compounds of the invention may, for example, be substantially free of other isomers or may, for example, be racemic mixtures or mixtures with all other stereoisomers or other selected stereoisomers. The chiral centers of the compounds identified by the present invention may have the S or R, or D or L configuration as defined in the IUPAC 1974 Recommendation (Recommendation).

In an eighth embodiment, the invention relates to pharmaceutical compositions comprising ACTH analogs and their administration to a subject in a manner that treats symptoms associated with ACTH-related disorders. The route of administration may be selected in accordance with known methods. ACTH analogs can be used as part of a pharmaceutical composition that includes a pharmaceutically acceptable carrier. Pharmaceutical compositions containing at least one ACTH analog of the invention may be formulated to treat diseases or disorders associated with elevated levels of ACTH in accordance with techniques well known in the pharmaceutical formulation arts, in admixture with conventional solid or liquid carriers or diluents and pharmaceutical additives (e.g., excipients, binders, preservatives, stabilizers, flavoring agents, etc.) suitable for the intended mode of administration.

Pharmaceutical compositions comprising ACTH analogs can be administered by any suitable means. For example, compositions comprising ACTH analogs can be administered by subcutaneous, intravenous, intramuscular, intracisternal injection, or infusion techniques (e.g., as a sterile injectable aqueous or nonaqueous solution or suspension); or intranasally, e.g., by inhalation spray; or topically, e.g., in the form of a cream or ointment; and is administered in a dosage unit formulation comprising a non-toxic pharmaceutically acceptable carrier or diluent. ACTH analogs of the invention can be administered, for example, in a form suitable for immediate release or extended release. Immediate release or extended release can be achieved by using suitable pharmaceutical compositions comprising ACTH analogs of the invention, or, particularly for extended release, by using devices such as subcutaneous implants or osmotic pumps. ACTH analogs of the invention can also be administered in the form of liposomes.

Pharmaceutical compositions comprising the ACTH analog polypeptides of the invention can be formulated according to known methods whereby their ACTH analog products are combined with a pharmaceutically acceptable carrier vehicle. Compositions useful for prophylactic and therapeutic treatments include one or more ACTH analog compounds and a means of application (e.g., injectable carrier systems, intranasal or transdermal).

As used herein, "carrier" includes pharmaceutically acceptable carriers, excipients, or stabilizers that are non-toxic to the contacted cells or subject at the dosages and concentrations employed. The phrase "pharmaceutically acceptable carrier" as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting a chemical of interest from one organ or part of the body to another organ or part of the body. Each carrier must be "acceptable" (in the sense of being compatible with the other ingredients of the formulation), non-toxic to the patient, and substantially pyrogen-free. Some examples of materials that can be used as pharmaceutically acceptable carriers include: (1) sugars such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) polled kagacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) ethylene glycols, such as propylene glycol; (11) polyols such as glycerol, sorbitol, mannitol and polyethylene glycol; (12) esters such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic salt; (18) ringer's solution; (19) ethanol; (20) a phosphate buffer solution; and (21) other non-toxic compatible materials that may be used in pharmaceutical formulations. In certain embodiments, the pharmaceutical compositions of the present invention are non-pyrogenic, i.e., do not cause a significant increase in temperature when administered to a patient. Generally, rawA physiologically acceptable carrier is a water-soluble pH buffer solution. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids or amino acid derivatives, such as norleucine or ornithine; monosaccharides, disaccharides, and other sugars including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN^TM、PLURONICS^TMOr PEG. Any carrier for ACTH analogs can be made by conventional means. However, if an alcohol is used in the carrier, the ACTH analog should be in micelles, liposomes, or "reverse" liposomes to prevent denaturation of the ACTH analog. Similarly, when the ACTH analog is placed in a carrier that is heated or has been heated, the ACTH analog should be added after the carrier has cooled slightly to avoid heat denaturation of the ACTH analog. The carrier is preferably sterile. One or more ACTH analogs can be added to these materials in liquid form or in a lyophilized state, wherein the ACTH analog dissolves when it encounters a liquid.

Prior to or simultaneously with mixing the modified ACTH analog with the carrier system, the ACTH analog can be in a stabilized buffered environment to maintain a pharmacologically suitable pH range, e.g., between about 4.0 and about 9.0, including pH about 5.0, 6.0, 7.0, 8.0 or any pH therebetween at 0.05 intervals or multiple 0.05 intervals therebetween, e.g., pH values of 5.2, 6.5, 7.4, 7.5, and 8.5. Lyophilized formulations suitable for storage and therapeutic formulations in aqueous solution form are prepared by mixing the ACTH analog active ingredient with the desired purity, optionally with physiologically acceptable carriers, excipients and stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, a.ed. (1980)). The stabilizing buffer should allow the ACTH analog to perform optimally. The buffer may comprise a reducing agent, such as dithiothreitol. The stabilizing buffer may also be or include a metal chelating agent, such as ethylenediaminetetraacetic acid disodium salt, or it may also contain a phosphate or citrate phosphate buffer or any other buffer.

Effective amounts of the compounds employed in the present invention can be determined by one of ordinary skill in the art. The effective dosage ratio or effective amount of an ACTH analog will depend, in part, on whether the ACTH analog is used therapeutically or prophylactically, the duration of exposure of the recipient to the infectious bacteria, the size and weight of the individual, and other considerations within the scope of medical judgment. The duration of use of compositions comprising ACTH analogs also depends on whether prophylactic, where use can be hourly, daily, and weekly for shorter periods of time, or therapeutic, where more intensive composition regimens are required, such that use will last for hours, days, or weeks and/or on a daily basis or at regular intervals of each day. The dosage form employed should provide the minimum number of units for the shortest time (a minimum amount of time). The dosage of the pharmaceutical composition of the invention and the concentration of the drug of interest will vary depending on the particular use. For example, when the ACTH analogs of the invention are administered to a patient for the treatment of cushing's syndrome and preterm birth, exemplary doses for an adult human include about 0.001 to 100mg of the active compound per kg of body weight per day, which can be administered in a single dose form or in separate dose forms (e.g., 1-4 times per day). It will be understood that the specific dose level and frequency of dosage for any particular compound may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the species, age, body weight, general health, sex and diet of the patient, mode and time of administration, rate of excretion, drug combination and the severity of the particular disease.

Determination of the appropriate dosage or route of administration is within the skill of the ordinarily skilled practitioner. Animal experiments provide reliable guidance for determining an effective amount for treating humans. The effective amount of interspecies modeling can be performed according to The principles set forth In Mordenti, J. and Chappell, W. "The use of The identities scaling In The toxicodynamics" In The toxicodynamics and New Drug Development, Yacobi et al, Pergamon Press, New York 1989, pages 42-96. The concentration of ACTH analog activity units that provide an effective amount of the ACTH analog can range from about 10 units/ml to about 500,000 units/ml of liquid (liquid in the wet and moist environment of the nasal and oral passages), and can generally range from about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 units/ml to about 50,000 units/ml. Thus, representative values include about 200 units/ml, 300 units/ml, 500 units/ml, 1,000 units/ml, 2,500 units/ml, 5,000 units/ml, 10,000 units/ml, 20,000 units/ml, 30,000 units/ml, and 40,000 units/ml. More specifically, the time of exposure to active ACTH analog units can affect the concentration of active ACTH analog units/ml of interest. Formulations for in vivo administration are preferably sterile. This can be easily achieved by filtration through sterile filtration membranes before or after lyophilization and reconstitution. The therapeutic compositions herein are generally placed in a container having a sterile access port, such as an intravenous solution bag or vial with a stopper pierceable by a hypodermic injection needle.

Examples of pharmaceutical compositions comprising ACTH analogs include injectable compositions, compositions that provide sustained release of the ACTH analog over a period of time of interest, compositions for dermal administration, injectable gels, coatings for implantable medical devices (coatings), mucoadhesive compositions, or compositions for the nose or other inhalable compositions.

In some examples, ACTH analogs can be administered by intramuscular or intravenous injection. For example, ACTH analogs can be administered intramuscularly, intravenously, subcutaneously, subdermally, intradermally, or a combination thereof.

The injectable compositions preferably comprise a suitable carrier and one or more ACTH analog compounds. The carrier may be composed of distilled water, salt solution, albumin, serum, or any combination thereof. In the case of intramuscular injection as the mode of administration, an isotonic formulation may be used. Typically, additives used to maintain isotonicity include sodium chloride, dextrose, mannitol, sorbitol, and lactose. In some cases, isotonic solutions, such as phosphate buffered saline, are used. Stabilizers include gelatin and albumin. In some examples, a vasoconstrictor is added to the formulation. The pharmaceutical formulations provided are sterile and pyrogen-free. Generally, as mentioned above, intravenous injection is most suitable. Exemplary compositions for parenteral administration include injectable solutions or suspensions which may contain, for example, suitable, non-toxic, parenterally acceptable diluents and solvents such as mannitol, 1, 3-dibutanol, water, ringer's solution, isotonic sodium chloride solution, or other suitable dispersing or wetting agents and suspending agents, including synthetic mono-or diglycerides and fatty acids including oleic acid. Glycerol or glycerin (1, 2, 3 glycerol) is a commercially available carrier for pharmaceutical use. Glycerol or glycerin can be diluted in sterile water for injection, or sodium chloride for injection or other pharmaceutically acceptable water-soluble injections, and used at a concentration of 0.1 to 100% (v/v), 1.0 to 50%, or about 20%. DMSO is an aprotic solvent that can enhance penetration of many topically applied drugs. DMSO is diluted in sterile water for injection, or in sodium chloride for injection or other pharmaceutically acceptable water-soluble injections, and is used at a concentration of 0.1 to 100% (v/v). Vehicles may also include ringer's solution, buffer solutions, and dextrose solutions, particularly when preparing solutions for intravenous administration.

Prior to or simultaneously with the addition of the ACTH analog to the carrier system, it is desirable that the ACTH analog be in a stabilized buffered environment that maintains a suitable pH range. The stabilization buffer allows the ACTH analog to perform optimally. The buffer may be a reducing agent, such as dithiothreitol. The stabilizing buffer may also be or include a metal chelating agent, such as ethylenediaminetetraacetic acid disodium salt, or it may also contain a phosphate or citrate phosphate buffer. Buffers in the carrier can be used as an environment for stabilizing ACTH analogs.

The carrier may optionally contain minor amounts of additives such as substances that increase isotonicity and chemical stability. Such agents are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, succinate, acetic acid and other organic acids or salts thereof; antioxidants, such as ascorbic acid; low molecular weight (less than about 10 residues) polypeptides, such as polyarginine or tripeptide; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; glycine; amino acids such as glutamic acid, aspartic acid, histidine or arginine; monosaccharides, disaccharides, and other sugars including cellulose or its derivatives, glucose, mannose, trehalose, or dextrins; chelating agents, such as EDTA; sugar alcohols, such as mannitol or sorbitol; counterions, such as sodium; nonionic surfactants such as polysorbates, poloxamers or polyethylene glycols (PEGs); and/or pharmaceutically acceptable salts, e.g. NaCl, KCl, MgCl₂、CaCl₂And the like.

The term "pharmaceutically acceptable salts" refers to the relatively non-toxic inorganic and organic acid addition salts of the inhibitors. These salts can be prepared in situ during the final isolation and purification of the inhibitor, or by reacting the purified inhibitor in the form of the free base with a suitable organic or inorganic acid and isolating the salt thus formed. Representative Salts include-14 hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthenate, mesylate, glucoheptonate, lactobionate, and laurylsulfonate, among others (see, e.g., Berge et al, (1977) 'Pharmaceutical Salts', j.pharm.sci.66: 1-19). In other cases, inhibitors useful in the methods of the invention may contain one or more acidic functional groups and are therefore capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable bases. "pharmaceutically acceptable salts" in these instances refer to the relatively non-toxic inorganic and organic base addition salts of the inhibitors. Likewise, these salts can be prepared in situ during the final isolation and purification of the inhibitor, or by reacting the purified inhibitor in its free acid form with a suitable base (e.g., hydroxide, carbonate, bicarbonate of a pharmaceutically acceptable metal cation), with ammonia, or with a pharmaceutically acceptable organic primary, secondary or tertiary amine. Representative alkali or alkaline earth metal salts include lithium, sodium, potassium, calcium, magnesium, and aluminum salts and the like. Representative organic amines useful for forming base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine and the like (see, e.g., Berge et al, supra).

Injectable pharmaceutical formulations containing ACTH analogs may optionally include other therapeutic agents, including antibacterial agents, anti-inflammatory agents, antiviral agents, and local anesthetics. The local anesthetic comprises tetracaine, tetracaine hydrochloride, lidocaine hydrochloride, dyclonine hydrochloride, quinicaine hydrochloride, cinchocaine hydrochloride, butamben picrate and pramoxine hydrochloride. Exemplary concentrations for local anesthesia are about 0.025% to about 5% (by weight) of the total composition. Anesthetics such as benzocaine can also be used at preferred concentrations of about 2% to about 25% (by weight).

The pharmaceutical composition may also optionally comprise one or more additional bioactive agents to inhibit steroid hormone synthesis (i.e., inhibitors of enzymes that catalyze different stages of steroid hormone synthesis) at different levels, including those bioactive agents reviewed in j. steroid biochem, volume 5, page 501, (1974), which include the following: a) diphenylmethane derivatives, such as amphenon B (which inhibits steroid hormone synthesis during the 11-beta-, 17-and 21-hydroxylation stages of hydroxylase); b) pyridine derivatives (SU-c series), such as metirapon (which inhibits synthesis during the 11-beta-hydroxylation stage of the hydroxylase); c) substituted α, α -glutaramides, such as aminoglutethimide (which prevents the synthesis of pregnenolone from cholesterol by inhibiting 20- α -hydroxylase and C20, C22-lyase); d) steroids, such as nitrilo epiandrostane (3 β -substituted steroid-3 β -hydroxy-5-androsten-17-one), which inhibit 3 β -deoxy steroid hydroxylase-5.4-isomerase (Steroids, Vol.32, p.257); e) spironolactone family steroids which act as rapidly dissociating antimineralocorticoids (PNAS USA 71(4), page 1431-1435, (1974)); f) the synthetic steroid ZK91587, described as an antimineralocortin, showed specific binding properties to kidney (z. naturforsch., 45b, p. 711-715, (1990)) and hippocampal type I MR (Life Science, 59, p. 511-21, (1996)) but not to type II GR. Therefore, it is routinely used as a tool to study MR function in tissues containing both receptor systems.

ACTH analogs can optionally be administered with biologically active agents that specifically inhibit glucocorticoid interaction with hormone receptors, including: a) mifepristone (11 β, 17 β) -11- [ -4- (dimethylamino) phenyl ] -17-hydroxy-17- (1-propynyl) androst-4, 9-dien-3-one which interacts with glucocorticoid receptor to form a complex which is unable to initiate the glucocorticoid response mechanism (Annals of New-York Academy of Science, Vol. 761. 296, pp. 296-310 (1995)); said compounds are also known as tocolytics (RU38486 or RU 486); b) non-steroidal substances (J.Steroid biochem., Vol.31, p.481-492, (1988)), such as, for example, oripavine hydrochloride (a derivative of isoquinoline-1- (3.4-dioxibene zilidine) -6.7-dioxy-1, 2, 3, 4-tetrahydroquinoline) or acetolsalicic acid (Moskovskava medicina, 1990, "Receptor mechanisms of the glucocorticoid effect", V.P.Goldkov). Anti-glucocorticoids (e.g., mifepristone) have been used clinically to treat non-pituitary cushing's syndrome, which is not amenable to surgery. In the case of mifepristone (antiprogestin and antiglucocorticoid), high doses (up to 800 mg/day) are required. With the adoption of systemic application strategies to improve activity and reduce cross-reactivity and adverse side effects, impressive progress has been made in the development of anti-hormonal drugs with greater potency and selectivity, particularly in the field of anti-estrogens and anti-androgens.

The effective dose rate or amount of ACTH analog to be administered parenterally and the duration of treatment will depend, in part, on the severity of the infection, the weight of the patient, the duration of exposure of the recipient to the infectious bacteria, the severity of the infection, and a number of other variables. The composition can be used once to several times a day, and can be used for a short period or a long period. Use lasts days or weeks. The dosage form employed should provide the minimum number of units for the shortest time (a minimum amount of time). It is contemplated that the concentration of ACTH analog active units that can provide an effective amount or effective dose of the ACTH analog can range from about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 units/ml of composition to about 10,000,000 units/ml of composition, from about 1000 units/ml of composition to about 10,000,000 units/ml of composition, and from about 10,000 units/ml of composition to about 10,000,000 units/ml of composition. The amount of activity units per ml and the duration of exposure will depend on the type of infection and the amount of vector exposed to the ACTH analog. It should be remembered that ACTH analogs function better when in a liquid environment. Thus, the effectiveness of ACTH analogs is related in part to the amount of moisture contained in the carrier. The concentration of ACTH analog used for treatment depends on the number of bacteria in the blood and the blood volume.

When ACTH analogs are administered in vivo, normal doses are from about 10ng/kg to 100mg/kg of subject body weight per day or higher, or from about 1. mu.g/kg/day to 10 mg/kg/day, depending on the route of administration. Guidance regarding specific dosages and methods of delivery is also provided below and in the literature. It is anticipated that different formulations will be effective for different therapeutic compounds and different conditions, and administration targeted to one organ or tissue will necessarily be delivered in a different manner than to another organ or tissue.

Pharmaceutical solutions for infusion or injection may be prepared in a conventional manner, for example by adding preservatives such as p-hydroxybenzoate or stabilizers such as alkali metal salts of ethylenediamine tetraacetic acid, and transferring the solution to infusion containers, injection bottles or ampoules. Alternatively, the compound for injection may be lyophilized together with other ingredients or lyophilized separately, and dissolved in a buffer solution or distilled water as needed at the time of use. Non-aqueous vehicles such as fixed oils, liposomes, and ethyl oleate may also be used herein.

In addition, various methods can be used to aid transport of ACTH analogs across cell membranes. ACTH analogs can be transported in liposomes, where the ACTH analog is "inserted" into the liposome by known techniques. Likewise, ACTH analogs can be in an antichaking. ACTH analogs can also be pegylated, wherein the polyethylene glycol is attached to an inactive portion of the ACTH analog. Alternatively, hydrophobic molecules can be used to transport ACTH analogs across cell membranes. Finally, glycosylation of ACTH analogs can be used to target specific internalizing receptors on cell membranes.

Materials with controlled release capabilities are particularly suitable for certain treatment methods. ACTH analogs can be used in combination with carrier systems, such as polymers that allow for sustained release of the ACTH analog compound over a desired period of time. High molecular weight excipients, such as cellulose (avicel) or polyethylene glycol (PEG), may also be included in such formulations. Such formulations may also include excipients to aid mucosal adhesion, such as hydroxypropyl cellulose (HPC), hydroxypropyl methylcellulose (HPMC), sodium carboxymethyl cellulose (SCMC), maleic anhydride copolymers (e.g., Gantrez), and agents to control release, such as polyacrylic acid copolymers (e.g., Carbopol 934). Lubricants, glidants, flavoring agents, coloring agents and stabilizers may also be used for ease of lubrication and use.

ACTH analog compounds can be combined with sustained release carrier compositions described in Loughman, U.S. patent No. 6,893,645 (filed 7/15/2002 and issued 5/17/2005), which is incorporated herein by reference. Other examples of sustained and controlled release formulations and methods that may be combined with ACTH analogs include published U.S. patent application nos. US2005/0164927a1 (proposed by Cheung et al, 8/18 2004), US2003/0125528a1 (proposed by Hay et al, 9/27 2002), US2003/0211966a1 (proposed by Kubek et al, 10/21 2002), US2003/0148938a1 (proposed by rmasha et al, 2001, 11/7), and US2003/0181361a1 (proposed by shama et al, 3/31 2003), the disclosures of which are hereby incorporated by reference in their entireties. In some embodiments, the controlled release therapeutic compositions can include a controlled release polymer and an effective amount of an ACTH analog. The controlled release polymer may be a water swellable polymer, which optionally includes a desired level of cross-linking moieties. The controlled release polymer may have a desired level of water solubility or have minimal water solubility.

Polymeric thickeners that may be used include those known to those skilled in the art, such as hydrophilic gelling agents and hydroalcoholic gelling agents that are commonly used in the cosmetic and pharmaceutical industries. Hydrophilic gelling agents or hydroalcoholic gelling agents may include, for example(B.F.Goodrich，Cleveland，Ohio)、(Kingston Technologies，Dayton，N.J.)、(Aqualon，Wilmington，Del.)、(Aqualon, Wilmington, Del.) or(ISP Technologies, Wayne, N.J.). The gelling agent may comprise from about 0.2% to about 4% by weight of the composition. More specifically, forExamples of weight percent ranges for the composition may be between about 0.5% to about 2%, and forAndis in a range of between about 0.5% to about 4% by weight. For theAndranges between about 0.5% to about 4% by weight of the composition.

Is one of many crosslinked acrylic polymers, commonly known by the name carbomer. These polymers dissolve in water and form clear and slightly hazy gels when neutralized with caustic materials such as sodium hydroxide, potassium hydroxide, triethanolamine and other amine bases.Is a cellulosic polymer that disperses in water and forms a uniform gel when fully hydrated. Other gelling polymers include hydroxyethyl cellulose, cellulose gums, MVE/MA decadiene crosspolymer, PVM/MA copolymer, and combinations thereof.

Preservatives may also be used in the present invention and may comprise, for example, from about 0.05% to about 0.5% by weight of the total composition. The use of preservatives ensures that if the product becomes contaminated with microorganisms, the formulation will prevent or reduce the growth of microorganisms. Some preservatives that may be used in the present invention include methylparaben, propylparaben, butylparaben, p-chloroxylenol, sodium benzoate, DMDM hydantoin, 3-iodo-2-propylbutylcarbamate, potassium sorbate, chlorhexidine gluconate, or combinations thereof.

Sustained release administration of ACTH analogs is desirable in formulations having release characteristics suitable for treating any disease or condition for which administration of the ACTH analog is desired, in which case microencapsulation of the ACTH analog is contemplated. Preferred microencapsulated compositions suitable for use with ACTH analogs include those described in U.S. patent No. 6,475,507 to pellett et al (filed 11/6/2000) and 6,911,218 to ignatous et al (filed 4/20/1999), both of which are incorporated herein by reference in their entirety. Microencapsulation of recombinant proteins for sustained release has been carried out with human growth hormone (rhGH), interferon- (rhIFN-), interleukin-2, and MN rgp120 efforts. Johnson et al, nat. med., 2: 795-799 (1996); yasuda, biomed. ther, 27: 1221-1223 (1993); hora et al, Bio/technology.8: 755, 758 (1990); cleland, "Design and Production of Single Immunization Vaccines Using polylactic polyol microspheres systems," in Vaccine Design: the Subunit and Adjuvant Approach, edited by Powell and Newman (Plenum Press: New York, 1995), page 439462; WO 97/03692, WO 96/40072, WO 96/07399; and U.S. patent No. 5,654,010.

The sustained or controlled release carrier may be a "long-term" or "slow" release carrier (e.g., certain nasal sprays, polymers, or capsules) or a "short-term" or "fast" release carrier (e.g., mouthwashes), which will have or provide a lower concentration of active (ACTH analog) units/ml, but for a longer period of time; whereas "short-term" or "rapid" release carriers will have or provide higher concentrations of active (ACTH analog) units per ml, but for shorter durations. The number of active units per ml and the duration of contact will depend on the type of infection, whether the treatment is prophylactic or therapeutic, and other variables. Thus, the number of administrations will depend on the circumstances and may be 1-4 or more times per day for a period of from one day to several weeks. Infections can occur in the skin, and thus such compositions can also be formulated for topical application using well known vehicles, such as those described in U.S. patents 6,056,954 and 6,056,955. Micelles and multilamellar micelles can also be used to control the release of ACTH analogs.

Bioabsorbable polymers, such as poly (lactic-co-glycolic acid) (PLGA) polymers, can be used in sustained release formulations of these proteins because of their biocompatible and widely biodegradable nature. The degradation products of PLGA, lactic acid and glycolic acid, are rapidly cleared by the human body. In addition, the degradability of the polymer can be adjusted from months to years, depending on its molecular weight and composition. Lewis, "Controlled release of bioactive agents from cellulose/glycolide polymer," in: chansin and r. langer (Eds.), Biodegradable Polymers as Drul: delivery Systems (Marcel Dekker: New York, 1990), pages 1-41.

In some examples, the therapeutic composition comprises a mucoadhesive sustained release formulation and an ACTH analog. As disclosed and described, mucosal linings include, for example, the upper and lower respiratory tracts, eyes, mouth, nose, rectum, vagina, periodontal pockets, intestines and colon. From E.R.Squibb&Of CoIs an adhesive that combines pectin, gelatin and sodium carboxymethylcellulose in a viscous hydrocarbon polymer for adhesion to the oral mucosa. However, a physical mixture of such hydrophilic and hydrophobic components may eventually fall off. In contrast, the hydrophilic and hydrophobic domains in the present disclosure produce insoluble copolymers. U.S. patent No. 4,948,580, which is incorporated herein by reference, describes a bio-adhesive drug delivery system. The compositions include a lyophilized polymer mixture formed from a copolymer of poly (methyl vinyl ether/maleic anhydride) and gelatin dispersed in an ointment base such as mineral oil containing dispersed polyethylene. U.S. patent No. 5,413,792, incorporated herein by reference, discloses pastes such as formulations comprising (a) a paste-like base comprising polysiloxane and water-soluble polymeric material, which may be present in a weight ratio of 3: 6 to 6: 3, and (B) an active ingredient. U.S. Pat. No. 5,554,380 claims to include havingSolid and semi-solid bioadhesive oral drug delivery systems of the two-phase water-in-oil system. One phase comprises from about 25% to about 75% (by volume) of an inner hydrophilic phase and the other phase comprises from about 23% to about 75% (by volume) of an outer hydrophobic phase, wherein the outer hydrophobic phase consists of 3 ingredients: (a) an emulsifier, (b) a glyceride, and (c) a wax material. Some representative release materials for administering antimicrobial agents are described in U.S. patent No. 5,942,243, the disclosure of which is incorporated herein by reference. Dosage forms of the disclosed compositions can be prepared by conventional methods.

Compositions comprising ACTH analogs can be administered by nasal spray, nasal drops, nasal ointment, nasal wash, nasal injection, nasal tamponade, bronchial spray, and inhaler. When ACTH analogs are introduced directly through the use of nasal sprays, nasal drops, nasal ointments, nasal washes, nasal injections, nasal tamponades, bronchial sprays, oral sprays, and inhalers, ACTH analogs can be in liquid and gel forms, where the liquid acts as a carrier. Although useful in liquid delivery forms, dry, anhydrous modified ACTH analogs can be administered by inhaler and bronchial spray. Nasal sprays can be either long-lasting sprays or regular release sprays, and can be manufactured by methods well known in the art. Inhalers can also be used to allow ACTH analogs to reach the lower extremities of the bronchial passages (including the lungs). Exemplary compositions suitable for nasal aerosol or inhalation administration include solutions in saline, and also include, for example, benzyl alcohol or other suitable preservatives, absorption promoters to increase bioavailability, and/or other stabilizers or dispersants such as those known in the art.

Compositions suitable for transdermal administration of ACTH analogs can be formulated using a vehicle that delivers at least one ACTH analog to or through the skin. Modes of application of ACTH analogs include a variety of different types and combinations of carriers including, but not limited to, water-soluble liquids, alcohol-based liquids, water-soluble gels, lotions, ointments, water-insoluble liquid bases, mineral oil bases, mixtures of mineral oil and petrolatum, lanolin, liposomes, protein carriers such as serum albumin or gelatin, powdered cellulose carmel, and other combinations. Modes of delivery of carriers containing therapeutic agents include, but are not limited to, spreading, spraying, timed release pACTH, liquid absorbable swabs, and combinations thereof. ACTH analogs can be applied directly to the bandage or in one of the other carrier ways. The bandage may be a commercially available wet or dry bandage in which the ACTH analog is present in lyophilized form on the bandage. This method of application is most effective for treating infected skin. The carrier of the topical composition may comprise semi-solid and gel-like vehicles including polymeric thickeners, water, preservatives, surfactants and emulsifiers, antioxidants, sunscreens, and solvents or mixed solvent systems. U.S. Pat. No. 5,863,560(Osborne) discusses a number of different combinations of carriers that can facilitate skin and drug contact. Another composition for topical application includes a topical carrier, such as Plastibase (mineral oil gelled with polyethylene).

In one example, the present invention comprises a dermatological composition having about 0.5% to 10% carbomer and about 0.5% to 10% drug, wherein the drug is present in a dissolved and particulate state. The dissolved drug has the ability to cross the stratum corneum, while the particulate state does not. The gel is completely formed by adding an amine base, potassium hydroxide solution or sodium hydroxide solution. More specifically, the drug may include dapsone, which is an antibacterial agent with anti-inflammatory properties. An exemplary ratio of dapsone in particulate form to dapsone in dissolved form is 5 or less.

In another example, the present invention comprises about 1% carbomer, about 80-90% water, about 10% ethoxydiglycol (ethoxydiglycol), about 0.2% methylparaben, about 0.3% to 3.0% dapsone (including both particulate dapsone and dissolved dapsone), and about 2% corrosive substances. More specifically, the carbomer may comprise980 and the corrosive substance may comprise hydrogen hydroxideA sodium solution.

Method of treatment

ACTH analog compounds are useful for treating a variety of ACTH-related disorders. Methods of treatment include administering one or more ACTH analog compounds to reduce ACTH-induced adrenal hormone secretion rates, to lessen the effects of high levels of ACTH on a patient, or to block excess ACTH while maintaining healthy adrenal function. ACTH analog compounds are useful for treating diseases associated with ACTH levels, such as diseases that respond to modulation of ACTH receptors (e.g., MC-2R). ACTH analog compounds can be administered to treat disorders associated with modulation of ACTH levels, such as reducing the effects of high levels of ACTH on a patient while maintaining normal healthy adrenal function.

ACTH analog compositions are useful, for example, in the treatment of ACTH related disorders such as cushing's syndrome, impaired immune response due to excessive corticosteroid secretion, causing premature labor (e.g., through the hypothalamic-pituitary-adrenal axis), and related disorders. In one aspect, a plurality of ACTH analogs are prepared and administered to a patient to evaluate the induction of cortisone in vivo.

ACTH-producing tumors in the pituitary gland can be treated by administering ACTH analog compounds in conjunction with high-resolution MR pituitary images. Administration of ACTH analog compounds in conjunction with sinus sampling establishes pituitary-derived ACTH hypersecretion, localization and laterality of preoperative pituitary tumors (see Oldfield, E.W. et al, "petrochemical sampling with and without a corticotropin-releasing hormone for the differential diagnosis of curing's syndrome," N Engl J Med., 325: 897-905 (1991); and Findling, J.W. et al, Endocrinol ab MetClin North Am., 30: 729-47 (2001)). While 70% of pituitary microadenomas can be successfully resected via the sphenoid approach, the surgical "cure" rate for large adenomas is only about one third of that of central specialty patients (Mampalam, T.J. et al, Ann Intern Med., 109: 487-93 (1988)).

ACTH analog compounds can also be administered in tumor therapy, including combination therapy with the treatment of excessive ACTH secretion or with pituitary radiation therapy to inhibit tumor growth and hormone levels. The effects of radiation are not apparent for several days and are associated with eventual pituitary damage and dysfunction in most patients (Brada, M. et al, "The long-term efficacy of cognitive and radiotherapeutics in The control of cervical adenomas," Clin Endocrinol., 38: 571-8 (1993)). Hypercortisolism, excessive cortisol secretion, can be completely resolved by adrenalectomy (surgical removal of one or both adrenals), but this approach is not able to inhibit pituitary tumor growth and is also associated with other comorbidities (see Trainer, P.J. et al, "curing's syndrome: therapeutic direct at the acquired glands," Endocrinol Metal North Am., 23: 571-.

ACTH analog compounds can be administered in combination with medical therapy, for example, co-administration with Cyproheptadine, an anti-serotonergic drug that is used to inhibit ACTH secretion but is ultimately less effective and has been discontinued (Krieger, d.t. et al, "Cyproheptadine-induced diabetes of cushing diseases," N Engl J med.293: 893-6 (1975)). Although The antifungal drug, ketoconazole, inhibits cortisol biosynthesis of The adrenal gland, The drug does not inhibit The growth of pituitary tumors or The secretion of ACTH, and The specific hepatic damage limits its long-term use (see Sonino, N., "The use of ketoconazole as an inhibitor of stereo production," N Engl J Med., 317: 812-8 (1987)).

In the ninth month of pregnancy, considerable amounts of CRH are produced by the placenta and fetal membranes (see Frim, D.M. et al, "Characterisation and geographic regulation of cognitive hormone-degrading hormone messenger RNA in human placenta," J Clin invest, 82: 287-292(1988)), causing an increase in maternal peripheral blood CRH concentration, particularly after 30 weeks of pregnancy (see Sasaki, A. et al, "Immunorective cognitive hormone-degrading hormone in human placenta expression, labor, and delivery," J Clin endocrine method, 64: 224-229(1987) and gold, R.S. 1204 et al, "High of cognitive hormone-degrading hormone protein in human placenta, J. III. et al," clinical hormone-degrading hormone protein kinase, J. III. et al, "clinical hormone-III" III. J. III. D.M. 119 (1986). Recent studies have shown increased levels of CRH in the blood of preterm pregnant women (see Wolfe, C.D. A. et al, "Plasma Corticotropin Releasing Factor (CRF) in abnormal prediction," Br J Obstet Gynaecol., 95: 1003-.

ACTH analogs can be used in combination with anti-childbirth drugs or drugs that prevent childbirth to delay childbirth. Examples of anti-childbirth drugs include ritodrine and terbutaline, which are beta-sympathogenic. However, to be effective, these drugs must typically be administered prior to or at the beginning of labor, and if administered after the beginning of labor, the effectiveness or safety is unreliable. Although low levels of anti-childbirth drugs are sometimes administered (typically by infusion) continuously to pregnant women during the high risk phase of pregnancy (typically between about 28 and 34 weeks of pregnancy), the drugs have adverse side effects on kidney function, respiratory function, heart rate, and general muscle tissue health. The dose must generally be maintained below, for example, about 0.1 cc/hour (1mg/cc of terbutaline solution). The effect of these low doses in avoiding the occurrence of labour is inconsistent and difficult to quantify. Accordingly, there is a need for therapeutic agents (treatments) and methods for treating premature labor. More specifically, there is a need for new therapeutic agents and methods for treating preterm labor that do not present existing adverse side effects.

In one embodiment, ACTH analog compositions can be administered to reduce ACTH biosynthesis. The method of treatment may be combined with, or preferably replace, administration of a drug, such as metyrapone. Preferably, ACTH analog compositions can be administered to treat The development of cushing's disease that develops during Pregnancy, as described in j.r. linday and l.k.nieman (2005) "The hypothioacetamic-Pituitary-additional Axis in Pregnancy: changes in Disease Detection and Treatment, "Endocrine Reviews 26: 775-800. Treatment of the disease (usually to prolong pregnancy or to prepare delivery) may involve administration of ACTH polypeptide and/or metyrapone, which is observed to be well tolerated without adverse effects on maternal liver function and fetal development. Metyrapone may be administered to a patient to reduce ACTH biosynthesis. While it is believed that the cause of corticosteroid hypersecretion is due to elevated ACTH levels (presumably from pituitary tumors in these women), ACTH analog compositions are particularly preferred as part of a strategy to reduce corticosteroid synthesis.

In another aspect, methods of treating a veterinary subject, such as methods for reducing stress hormones to facilitate healthy growth of high-density agricultural and aquaculture species, are provided. Compositions comprising one or more ACTH analog compounds can be administered in a manner that reduces stress hormones to facilitate healthy growth of high-density agricultural and aquaculture species.

Examples

The following examples serve to illustrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the materials and techniques disclosed in the examples which follow, given the disclosure of the inventors, may be readily utilized in the practice of the present invention and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Example 1 serum corticosteroid Induction assay (in vivo)

ACTH analogs having reduced ACTH-mediated corticosteroid secretion are identified by measuring the level of corticosteroid in the blood of a subject following administration of the ACTH analog by performing an in vivo serum corticosteroid induction assay.

In vivo serum corticosteroid induction assays were performed according to the following method. First, we bred male FVB/N mice of 2-3 months of age (5 per group) were injected intraperitoneally with dexamethasone (0.4 mg/0.1ml PBS per mouse; Sigma, St. Louis, Mo.) to suppress their endogenous ACTH production. See Hajos, GT et al, "students of the potency of polypeptides with action by a new method based on compositions of microorganisms," Steroids Lipids Res 3: 225-228 (1972); costa, JL et al, "statistical analysis of empirical consistent ACTH residues," Gen Comp Endocrinol, 136: 12-16 (2004); and Karpac, J et al, "Development, Maintenance, and Function of the additional glass and in Early post natal Pro-ocular in Null music Mice," Endocrinology (2005), published online before 24/2/2005, all of which are incorporated herein by reference.

Second, after 90 to 120 minutes, mice inhibited with dexamethasone were injected either with unmodified mACTH (1-24), or one of several ACTH analog peptide compounds (1 μ g/0.1mL PBS/0.5% BSA), or vehicle only (control) (0.1mL PBS/0.5% BSA), subcutaneously between the scapulae. The following ACTH analog peptide compounds were administered to each mouse: 1: murine ACTH 1-24; 2: V26F, E30K; 3: P19W, K21E, Y23R; 4: E30K, P36R; 5: ALA 19-24; 6: V26F, P36R; 7: P19W, K21E; 8: P19W, K21A, delY 23; 9: P19W, K21A; 10: KRRP16-19RAAW (AT814) (SEQ ID NO: 20).

Third, after 1 hour, blood was collected from the small incision in the tail within 1 minute, and serum was snap frozen and stored at-80 ℃ until analysis. By competitive radioimmunoassay method (¹²⁵I RIA kit; ICN, Costa Mesa, CA) according to the manufacturerThe recommendation of (a) determines serum corticosterone. For each sample analyzed, 1 μ l serum was used. Samples were analyzed in duplicate. The results described above for ACTH analog peptides are shown in the graph in fig. 1, with activity shown as a percentage of ACTH activity. These ACTH analogs have lower activity compared to native ACTH.

Example 2 serum corticosteroid inhibition assay (in vivo)

ACTH analogs having reduced ACTH-mediated corticosteroid secretion are identified by performing an in vivo serum corticosteroid inhibition assay to test their ability to inhibit adrenal hormone production induced by unmodified ACTH. After administering the ACTH analog test compound in combination with a compound having known corticosteroid-inducing activity (e.g., ACTH or another ACTH analog), the level of corticosteroid in the subject's blood is measured. ACTH analog test compounds may be administered prior to, concurrently with, or subsequent to administration of the corticosteroid-producing compound.

An in vivo serum corticosteroid inhibition assay was performed as follows. ACTH analogs test compounds, such as those in table 1, having low activity, were tested for their ability to inhibit adrenal hormone production induced by unmodified ACTH.

First, we bred male FVB/N mice of 2-3 months of age (5 per group) were injected intraperitoneally with dexamethasone (0.4 mg/0.1ml PBS per mouse; Sigma, St. Louis, Mo.) to suppress their endogenous ACTH production.

Second, after 90 to 120 minutes, animals were injected subcutaneously between the scapulae with wild-type mACTH and/or test ACTH analog peptide (1 μ g/0.1mL PBS/0.5% BSA) or vehicle only (0.1mL PBS/0.5% BSA).

Third, after 1 hour, blood was collected from the small incision in the tail within 1 minute, and serum was snap frozen and stored at-80 ℃ until analysis. By competitive radioimmunoassay method (¹²⁵I RIA kit; ICN, Costa Mesa, CA) determined blood according to manufacturer's recommendationsQingcorticosterone. For each sample analyzed, 1 μ l serum was used. Samples were analyzed in duplicate.

The results described above for the analog AT814(SEQ ID NO: 20) are shown graphically in FIG. 2. The activity of the peptide is shown as a percentage of corticosterone induction, where native mouse ACTH is set to 100%. Comparison of corticosterone induction 1 hour after the last peptide injection showed that AT814 reduced corticosterone induction to 35% of wild-type ACTH when used 30 minutes prior to ACTH injection; when used concurrently with ACTH injection, AT814 reduced corticosterone induction to 10% of wild-type ACTH.

EXAMPLE 3 Addrecombination assay (in vitro)

ACTH analogs having the ability to bind to adrenoreceptors can be identified by performing in vitro adrenal binding assays. The amount of radiolabeled ACTH analog test compound bound to explanted adrenal membrane can be determined to identify ACTH analogs having adrenoreceptor binding activity. Preferably, this can be done in serum-free medium (in vitro serum-free adrenal binding assay).

ACTH analogs that have the ability to bind to an adrenal receptor with a higher binding affinity than compounds known to have adrenal binding activity (preferably ACTH) can also be identified by an in vitro adrenal competitive binding assay in which the decrease in the amount of radiolabeled compound that binds to the adrenal gland (e.g., ACTH) is measured by measuring the different concentrations of non-radiolabeled ACTH analog test compound in media containing explanted adrenal membranes. The reduction in adrenal membrane binding by radiolabeled adrenal binding compounds can be used to identify and characterize the binding activity of ACTH analog test compounds. The decrease in binding of radiolabeled ACTH was determined in a series of in vitro adrenal competitive binding assays by adding ACTH analog test compounds to adrenal membrane medium.

In vitro serum-free adrenal competition binding assays were performed as follows. First, the adrenal glands were removed from the mice, cut out of fat and cut in half. See Costa, JL et al, "microbiological analysis of empirical consistent ACTH residues," Gen Comp endocrinol.136: 12-16 (2004); and Weber, MM et al, "Postnatal overexpression of insulin-like growth factor II in transgenic mice is associated with artificial with advanced polymorphism, and" Endocrinology 140: 1537-1543(1999), which is incorporated herein by reference.

Second, each adrenal half was placed in each air in a 4-well dish, and the adrenal glands of each mouse were placed in one dish. The two halves of adrenal gland were incubated in 0.5ml serum free medium (M199; Invitrogen) at 5% CO²Equilibrate at 37 ℃ for 30 minutes.

Third, the two halves of the adrenal glands were macerated or split using a Dounce homogenizer to form the membrane fraction. This fraction was resuspended in the appropriate buffer (Tris or HEPES). Then adding to the resuspended membrane fraction¹²⁵I radiolabelled ACTH (200ml) and non-radiolabelled test compounds (e.g. ACTH or ACTH analogues such as AT814(SEQ ID NO: 20)) were added in amounts such that the total concentration of test compounds was 0nm, 10nm, 100nm and 1000 nm; no peptide was added to the control. The membrane fraction was recovered and the radioactive signal was detected using a gamma detector. The decrease in the detected radioactive signal after addition of the non-radiolabeled test compound can be correlated with the binding affinity of a variety of test compounds. The decrease in the radioactive signal after addition of the ACTH analog peptide test compound indicates that it has increased affinity for adrenal ACTH receptors such as MC-2R and has the ability to displace ACTH at adrenal MC-2R receptors.

Figure 3 shows the results of a series of in vitro serum-free adrenal competition binding assays.

EXAMPLE 4 adrenal suppression assay (in vitro)

ACTH analogs that reduce and block corticosteriod secretion by ACTH-mediated adrenal membranes were identified by performing an in vitro serum-free corticosteroid inhibition assay to test their ability to inhibit adrenocorticotropic hormone production induced by unmodified ACTH. Corticosteroid levels are measured in serum-free media after combined administration of an ACTH analog test compound with a compound having known corticosteroid-inducing activity (e.g., ACTH or another ACTH analog). ACTH analog test compounds may be administered prior to, concurrently with, or subsequent to administration of the corticosteroid-producing compound.

The in vitro serum-free adrenal suppression assay was performed as follows. First, the adrenal glands were removed from the mice, cut out of fat and cut in half. See Costa, JL et al, "microbiological analysis of empirical consistent ACTH residues," Gen Comp endocrinol.136: 12-16 (2004); and Weber, MM et al, "Postnatal overexpression of in sulin-like growth factor II in transgenic mice associated with biochemical hyperplasia and enhanced sterogenesis," Endocrinology 140: 1537-1543(1999), which is incorporated herein by reference.

Second, each adrenal half was placed in each air in a 4-well dish, and the adrenal glands of each mouse were placed in one dish. The two halves of adrenal gland were incubated in 0.5ml serum free medium (M199; Invitrogen) at 5% CO²Equilibrate at 37 ℃ for 30 minutes.

Third, the medium was removed and replaced with 0.5ml of M199 containing ACTH, AT814 or both (100 ng/ml each); no peptide was added to the control.

Fourth, after 2 hours of incubation, the medium was removed, 4 adrenal halves from each mouse were pooled and analyzed by corticosterone using standard RIA methods. By competitive radioimmunoassay method (¹²⁵I RIA kit; ICN, Costa Mesa, CA) corticosterone was determined according to the manufacturer's recommendations. For each sample analyzed, 1 μ l serum was used. Samples were analyzed in duplicate. The results are shown in FIG. 4.

Example 5 (predictive) therapeutic veterinary administration

Fish hatching presents a number of challenges because the goal of maximizing the total tonnage of fish for commercial purposes is disrupted by the effects of crowding and infection transmission. In these environments, fish attempt to address this chronic stress through the hypothalamic/pituitary/interrenal (HPI) axis. In these cases, mortality, desensitization to corticosteroids, and development of new homeostasis for population density result at some level from the increase in corticosteroids. In contrast, during this "regulatory" cycle, long-term increases in corticosteroid levels reduce resistance to infection.

The HPI of hatchery fish can be adjusted by administering ACTH analogs of the invention via timed release, for example from silicone rubber capsule implants in these fish. Such buffering of the HPI axis should improve survival and possibly facilitate weight gain in hatchery fish.

Fish implanted with ACTH analogs of the invention or ACTH alone may be placed under environmental conditions that activate the HPI axis (i.e., crowding, pH changes, increased ammonia and nitrate). Two parameters that can be analyzed in these examples are mortality and weight change. Such parameters may demonstrate whether ACTH analogs of the invention are effective in reducing corticosterone levels in fish.

Claims (38)

1. A composition comprising an isolated ACTH analog peptide, or a pharmaceutically acceptable salt thereof, wherein the ACTH analog peptide has the formula:

A1-A2-A3-A4-A5-A6-A7-A8-A9-A10-A11-A12-A13-A14-A15-A16-A17-A18-A19-A20-A21-A22-A23-A24-Asn-A26-Ala-Glu-A29-A30-Ser-Ala-Glu-Ala-Ph e-A36-Leu-Glu-Phe-R

wherein:

a1 is Ser or D-Ser;

a2 is Tyr or D-Tyr;

a3 is Ser or D-Ser;

a4 is Met, D-Met or Nle;

a5 is Glu, D-Glu, Cys or Asp;

a6 is His or D-His;

a7 is Phe, D-Phe, D-p-iodo-Phe or D-1-napth-Ala;

a8 is Arg or D-Arg;

a9 is Trp or D-Trp;

a10 is Gly, Cys, Lys, Orn, Dab or Dpr;

a11 is Lys, D-Lys or Gly;

a12 is Pro or D-Pro;

a13 is Val or D-Val;

a14 is Gly or D-Gly;

a15 is Lys, Arg, Ala, Gly, Val, Leu, Ile, Gln, Asn, Glu, Asp, Nle, or an amino acid having an alkyl side chain;

a16 is Lys, Arg, Ala, Gly, Val, Leu, Ile, Gln, Asn, Glu, Asp, Nle, or an amino acid having an alkyl side chain;

a17 is Lys, Arg, Ala, Gly, Val, Leu, Ile, Gln, Asn, Glu, Asp, Nle, or an amino acid having an alkyl side chain;

a18 is Lys, Arg, Ala, Gly, Val, Leu, Ile, Gln, Asn, Glu, Asp, Nle, or an amino acid having an alkyl side chain;

a19 is Pro, Trp, or Ala;

a20 is Val or Ala;

a21 is Lys, Ala Pro or Glu;

a22 is Val or Ala;

a23 is Tyr, Ala, Arg or deleted;

a24 is Pro or Ala;

a26 is Gly, Val or Phe;

a29 is Asp or Asn;

a30 is Glu or Lys; and is

A36 is Pro or Arg;

wherein the content of the first and second substances,

a) r is OH or a pharmaceutically acceptable salt moiety; and is

b) Lys and Arg are not adjacent amino acids at positions 16-18; and is

c) The amino acid residues at positions 15-19 are not Ala-Lys-Ala-Arg-Trp, Lys-Ala-Arg-Ala-Trp, Ala-Ala-Ala-Arg-Pro, Arg-Ala-Ala-Ala-Pro, or Ala-Ala-Ala-Pro; and is

d) Amino acid residues at positions 25-39 are optionally deleted; and is

e) The peptide is not Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-Gly-Lys-Arg-Ala-Ala-Trp-Val-Lys-Val-Tyr-Pro-Ac (SEQ ID NO: 20).

2. The composition of claim 1 comprising an isolated ACTH analog peptide or pharmaceutically acceptable salt thereof, wherein the one or more amino acid substitutions of residues 16-18 do not include any two adjacent Lys or Arg amino acid residues.

3. The composition of claim 1, wherein the ACTH analog peptide comprises at least one Ala and at least one Arg residue substituted at any two of amino acid positions 15, 16, 17, or 18.

4. The composition of claim 1, wherein the amino acid at position 15 is selected from the group consisting of: lys, Ala and Gln; and positions 16, 17 and 18 do not include any two adjacent Lys or Arg amino acid residues.

5. The composition of claim 1, wherein administration of the ACTH analog peptide reduces ACTH-induced corticosteroid secretion by at least 10% in an in vivo serum corticosteroid inhibition assay.

6. The composition of claim 1, wherein the ACTH analog peptide binds to an adrenal membrane and displaces the sequence of SEQ ID NO: 2, wherein the binding of said peptide is determined by an in vitro serum-free adrenal competition binding assay.

7. The composition of claim 6, wherein the ACTH analog peptide is encoded by a sequence that is greater than SEQ ID NO: 2 binds MC-2R adrenal membrane with at least two-fold higher affinity.

8. The composition of claim 2, wherein the ACTH analog peptide reduces corticosterone production by the adrenal membrane induced by ACTH in an in vitro serum-free adrenal suppression assay.

9. The composition of claim 1, wherein

A19 is Pro, Trp, or Ala;

a26 is Gly, Val or Phe;

a30 is Glu or Lys; or

A36 is Pro or Arg;

wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 2 causes at least a 10% reduction in ACTH-induced corticosterone production in an in vitro serum corticosteroid-induced assay as compared to ACTH-induced corticosterone production in the adrenal membrane by the ACTH analog.

10. The composition of claim 9, wherein positions 16, 17 and 18

a) Does not include any two adjacent Lys or Arg amino acid residues; and are

b) Selected from: lys, Arg, Ala, Gly, Val, Leu, Ile, amino acid analogs comprising alkyl side chains, Nle, Gln, Asn, Glu, and Asp.

11. The composition of claim 9, wherein amino acid residues 25-39 are deleted.

12. The composition of claim 1 comprising an isolated ACTH analog peptide, or a pharmaceutically acceptable salt thereof, wherein the ACTH analog peptide has the formula:

Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-Gly-Lys-A16- A17-A18-A19-A20-A21-A22-A23-A24-Asn-A26-Ala-Glu-A29-A30-Ser-Ala-Glu-Ala-Phe-A36-Leu-Glu-Phe-R

wherein:

a16 is Lys or Arg;

a17 is Lys or Ala;

a18 is Lys or Ala;

a19 is Pro, Trp, or Ala;

a20 is Val or Ala;

a21 is Lys, Ala, Pro or Glu;

a22 is Val or Ala;

a23 is Tyr, Ala, Arg or deleted;

a24 is Pro or Ala;

a26 is Gly, Val or Phe;

a29 is Asp or Asn;

a30 is Glu or Lys; and is

A36 is Pro or Arg;

wherein the content of the first and second substances,

a) r is OH or a pharmaceutically acceptable salt moiety; and is

b) Lys and Arg are not adjacent amino acids at positions 16-18; and is

c) Amino acid residues at positions 25-39 are optionally deleted; and is

d) The peptide is not Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-Gly-Lys-Arg-Ala-Ala-Trp-Val-Lys-Val-Tyr-Pro-Ac (SEQ ID NO: 20).

13. The composition of claim 12, wherein the amino acid at position 19 is Trp.

14. The composition of claim 12, wherein the ACTH analog peptide comprises at least one Ala and at least one Arg residue substituted at any two of amino acid positions 15, 16, 17, or 18.

15. The composition of claim 12, wherein administration of the ACTH analog peptide reduces ACTH-induced corticosteroid secretion by at least 10% in an in vivo serum corticosteroid inhibition assay.

16. The composition of claim 12, wherein amino acid residues 25-39 are deleted.

17. A method for manufacturing a medicament for treating ACTH-related disorders comprising the step of admixing an ACTH analog according to any one of claims 1-16 with a suitable carrier.

18. The method of claim 17, wherein one symptom of ACTH-related disorder is elevated blood corticosteroid concentrations.

19. The method of claim 17, wherein the ACTH-related disorder is selected from the group consisting of: cushing's disease, premature labor, pituitary tumors, and hypothalamic/pituitary/inter-renal (HPI) axis disease.

20. The method of claim 17, wherein the medicament is formulated for administration by injection, inhalation, or transdermal absorption.

21. A method of making a medicament for treating ACTH-related disorders, comprising administering a peptide according to SEQ id no: 20 with a suitable carrier.

22. Use of an ACTH analog as described in any one of claims 1-16 in the preparation of a pharmaceutical composition for treating an ACTH-related disorder in a subject in need thereof.

23. The use of claim 22, wherein the pharmaceutical composition is formulated for administration to a subject by injection, inhalation, or transdermal absorption.

24. The use of claim 23, wherein the pharmaceutical composition is formulated for administration by injection, wherein the treatment further comprises the step of injecting the pharmaceutical composition into the subject.

25. The use of claim 22, wherein the treatment further comprises the step of determining the subject's blood corticosteroid level prior to administration of the pharmaceutical composition.

26. The use of claim 24, wherein the pharmaceutical composition is injected subcutaneously or intravenously.

27. The use of claim 24, wherein the treatment further comprises the step of administering a drug that reduces the subject's blood corticosteroid level prior to injecting the pharmaceutical composition into the subject.

28. The use of claim 27, wherein the medicament that reduces blood corticosteroid levels comprises dexamethasone.

29. The use of claim 22, wherein the subject is a human.

30. The use of claim 22, wherein the pharmaceutical composition comprises a sustained release composition.

31. According to SEQ ID NO: 20 for use in the preparation of a pharmaceutical composition for treating an ACTH-related disorder in a subject in need thereof.

32. ACTH analog compounds were screened to identify compounds that are similar to SEQ ID NO: 2 which induces less corticosteroid secretion from the adrenal membrane than does the unmodified ACTH, comprising the step of contacting the adrenal membrane with the ACTH analog.

33. The method of claim 32, wherein the screening method further comprises the steps of:

a. providing a first adrenal membrane and a second adrenal membrane;

b. contacting a first adrenal membrane with a polypeptide comprising a sequence according to SEQ ID NO: 2 and then determining the presence of a second composition of unmodified peptides according to SEQ ID NO: 2 by a first concentration of corticosteroid secreted by a first adrenal membrane after contact;

c. a second adrenal membrane is contacted with a second composition comprising an ACTH analog, and a second concentration of corticosteroid secreted by the second adrenal membrane after contact with the ACTH analog is determined.

34. The method of claim 33, further comprising the step of comparing the concentration of the first secreted corticosteroid to the concentration of the second secreted corticosteroid.

35. The method of claim 34, further comprising the step of determining whether the second compound induces less corticosteroid secretion than the first compound.

36. The method of claim 32, wherein the ACTH analog is a polypeptide according to SEQ id no: 20 ACTH analog.

37. The method of claim 32, wherein the first and second adrenal membranes are located in the subject and the concentration of the corticosteroid is the measured concentration in the blood of the subject.

38. The method of claim 32, wherein the first and second adrenal membranes are removed from the subject and the corticosteroid concentration is a concentration determined in serum-free media.