RU2286995C2 - Agonists of corticotropin-releasing factor receptor - Google Patents

Abstract

FIELD: medicine, peptides.

SUBSTANCE: invention relates to biologically active compounds representing isolated derivatives of corticotropin-releasing factor. Invention presents non-native peptide of the formula: I-V-L-S-L-D-V-P-I-G-L-L-Q-I-L-L-E-Q-X₁₉-K-X₂₁-X₂₂-X₂₃-X₂₄-X₂₅-X₂₆-Q-A-T-T-N-A-R-I-L-A-R-V (SEQ ID NO: 531) wherein (a) X₁₉ is chosen from the group consisting of D and E; (b) X₂₁ is chosen from the group consisting of A and Q; (c) X₂₂ is chosen from the group consisting of R, E and K; (d) X₂₃ is chosen from the group consisting of A, K and N; (e) X₂₄ is chosen from the group consisting of A, E and L; (f) X₂₅ is chosen from the group consisting of R and K; (g) X₂₆ is chosen from the group consisting of E and Q. Isolated derivatives of corticotropin-releasing factor and nucleic acids encoding thereof are effective agents used in treatment of disorders modulated by corticotropin-releasing factor-2, such as muscular dystrophy.

EFFECT: valuable medicinal properties of agonists.

Description

FIELD OF THE INVENTION

This invention relates to the use of new peptides and nucleic acids encoding them for the treatment of disorders modulated by CRF2R.

CROSS RELATIONS TO RELATED APPLICATIONS

Priorities for this application are U.S. provisional application 60/349117, interim application filed January 16, 2002, US provisional application 60/376337, filed April 29, 2002, US provisional application 60/388895 filed June 14, 2002, and US provisional application 60/411988 , subject September 19, 2002, each of which is hereby incorporated by reference in its entirety.

BACKGROUND

CRFR and ligands

There are at least two corticotropin releasing factor receptors (CRF ₁ R and CRF ₂ R) identified so far that belong to the class of G-protein coupled receptors (GPCR). Activation by agonists of CRF ₁ R or CRF ₂ R leads to Gαs activation of adenylate cyclase. Adenylate cyclase catalyzes the formation of cAMP, which in turn has multiple effects, including activation of protein kinase A, intracellular calcium release and activation of mitogen-activated protein kinase (MAP kinase). The increased synthesis of intracellular inositol triphosphate shown in other studies after activation by CRF receptor agonists suggests that CRFRs are also associated with Gαq.

The human, rat, mouse, chicken, cow, catfish, frog and sheep CRF ₁ R and CRF ₂ R cloned. Each of CRF ₁ R and CRF ₂ R has a unique distribution pattern. Three isoforms of the CRF ₂ R receptor, human alpha, beta and gamma, were cloned. Homologs of alpha and beta CRF ₂ R identified in rats.

Several CRFR ligands / agonists are known, and these include corticotropin releasing factor (or hormone, CRF, CRH), urocortin I, urocortin II (or a peptide related to stressoscopy), urocortin III (or stressoscope), urotensin I, sauvagin and other related peptides. Corticotropin releasing factor binds and activates CRF ₁ R and CRF ₂ R. CRF is the main modulator of body reactions to stress. The specified 41-amino acid peptide controls many nervous, endocrine and immune processes as the primary regulator of the hypothalamic-pituitary-adrenal hormonal axis (HPA axis). In addition, there is significant sequence homology between all known CRFR ligands. Moreover, two selective CRF ₂ R ligands have been identified, urocortin II (or the peptide related to streskopin) and urocortin III (streskopin). These peptides have been identified in many species of mammals and fish.

CRF receptors can be distinguished pharmacologically from non-CRFR using selective agonists and receptor antagonists. These selective agonists and antagonists, as well as CRFR knocked out mice, were used to determine which CRF receptor mediates a particular biological response.

The role of CRF ₁ R is pretty well established. Mice in which the CRF ₁ R gene is disrupted (knock-out of CRF ₁ R), exhibited attenuated stress reaction and attenuated behavior similar alarm state. CRF ₁ R is the main mediator of the HPA axis. In particular, CRF, which is released from the hypothalamus and transported to the anterior pituitary gland through the hypothalamic-pituitary portal system, interacts with CRF ₁ R present on cells located in the anterior pituitary gland. Activation of CRF ₁ R by agonists leads to the release of ACTH from the cells of the anterior pituitary into the circulatory system. Released ACTH binds to the ACTH receptor present on cells located in the adrenal cortex, leading to the release of adrenal hormones, including corticosteroids. Corticosteroids mediate many effects, including, but not limited to, suppressing the immune system through a mechanism that involves atrophy of the thymus and spleen. Thus, activation of CRF ₁ R indirectly leads to downregulation of the immune system through activation of the HPA axis.

The role of CRF ₂ R is worse. Mice in which the gene was disrupted CRF ₂ R (knockout of CRF ₂ R), exhibit attenuated or reduced food intake following stimulation with urocortin, lack of vasodilation, but a normal stress response. Experiments with CRF ₂ R showed that CRF ₂ R is responsible for the hypotensive / vasodilating effect of CRFR agonists and for the reduction in food intake observed after treatment of mice with CRFR agonists.

Atrophy and hypertrophy of skeletal muscle

In addition, CRF ₂ R is involved in modulating skeletal muscle atrophy and induction of hypertrophy. Skeletal muscle is a plastic tissue that easily adapts to changes in either the physiological needs for work or in metabolic needs. Hypertrophy refers to an increase in skeletal muscle mass, while skeletal muscle atrophy refers to a decrease in skeletal muscle mass. Acute skeletal muscle atrophy can be caused by several reasons, including but not limited to: cessation of use due to surgery, bed rest, or bone fracture; denervation / nerve damage due to damage to the spinal cord, autoimmune disease or infectious disease; use of glucocorticoids in case of unrelated conditions; sepsis as a result of infection or for other reasons; limitation of nutrients due to illness or starvation; and space flights. Atrophy of skeletal muscles occurs during normal biological processes, however, in some medical cases, the specified normal biological process leads to a level of muscle atrophy that weakens health. For example, acute skeletal muscle atrophy provides a significant limitation in the rehabilitation of patients from immobilization, including, but not limited to, immobilization accompanying the orthopedic procedure. In such cases, the rehabilitation period required to eliminate skeletal muscle atrophy is often much longer than the period of time required to restore the initial damage. Such acute atrophy in the event of discontinuation of use is a particular problem for an elderly person who may already suffer from a significant age-related deficiency of muscle function and mass, since such atrophy can lead to persistent disability and premature mortality.

Skeletal muscle atrophy can also result from chronic conditions such as malignant cachexia, chronic inflammation, AIDS related cachexia, chronic obstructive pulmonary disease (COPD), congestive heart failure, genetic diseases such as muscular dystrophy, neurodegenerative diseases, and sarcopenia ( age-related loss of muscle mass). In the case of these chronic conditions, skeletal muscle atrophy can lead to premature loss of mobility, added to the incidence of the disease.

Little is known about the molecular processes that control skeletal muscle atrophy or hypertrophy. Despite the fact that the triggering mechanism for the initiation of skeletal muscle atrophy is different for different events initiating atrophy, several common biochemical changes occur in the affected skeletal muscle fiber, including decreased protein synthesis and increased protein degradation and changes in isoforms of both contractile proteins and metabolic proteins enzymes characteristic of switching from slow fibers (high oxidative metabolism / slow isoforms of contractile proteins) to fast fibers (high glycolytically th metabolism / fast isoforms of contractile proteins). Additional changes in skeletal muscle that occur include vascular loss and remodeling of the extracellular matrix. Atrophy is observed in both fast and slow muscles under appropriate conditions, while the relative loss of muscle mass depends on the specific stimuli of atrophy or conditions. It is important that all these changes are coordinated and turned on and off, depending on changes in physiological and metabolic needs.

The processes by which atrophy or hypertrophy occurs are conservative among mammalian species. Many studies have shown that with atrophy, both rodents and humans have the same basic molecular, cellular, and physiological processes. Thus, models of skeletal muscle atrophy in rodents have been successfully used to understand and predict atrophic reactions in humans. For example, atrophy induced in many ways in rodents and humans results in similar changes in muscle anatomy, cross-sectional area, function, fiber type switching, expression of contractile proteins and histology. In addition, several agents have been shown to regulate skeletal muscle atrophy in both rodents and humans. These agents include anabolic steroids, growth hormone, insulin-like growth factor I, beta-adrenergic agonists and CRF ₂ R agonists. These data taken together indicate that skeletal muscle atrophy is the result of common mechanisms in both rodents and person.

Although it has been shown that some agents regulate skeletal muscle atrophy and are approved for human use in case of such an indication, these agents have undesirable side effects, such as cardiac muscle hypertrophy, neoplasia, hirsutism and androgenization in women, increased morbidity and mortality, liver damage, hypoglycemia, musculoskeletal pain, increased tissue turgor, tachycardia and edema. Currently, there are no highly effective and selective methods for treating either acute or chronic skeletal muscle atrophy. Thus, there remains a need to identify other therapeutic agents that treat skeletal muscle atrophy.

Muscular dystrophy

Muscular dystrophy covers a group of hereditary, progressive muscle diseases that are clinically distinguished by the selective distribution of skeletal muscle weakness. The two most common forms of muscular dystrophy are Duchenne dystrophy and Becker dystrophy, each of which results from the inheritance of a mutation in the dystrophin gene, which is located at the Xp21 locus. Other dystrophies include, but are not limited to, Leiden muscular dystrophy resulting from mutations in multiple genetic loci, including the p94, adhaline, γ-sarcoglycan and β-sarcoglycan loci; facial-shoulder-shoulder muscular dystrophy (Landusi-Dejerine), myotonic dystrophy and Emery-Dreyfuss muscular dystrophy. Symptoms of Duchenne muscular dystrophy, which occurs almost exclusively in men, include duck gait, walking on tiptoe, lordosis, frequent falls, and difficulty standing and climbing stairs. Symptoms begin at about 3–7 years old, with most patients being wheelchair bound by 10–12 years old, and many die at about 20 years of age due to respiratory complications. Modern treatment for Duchenne muscular dystrophy consists in the introduction of prednisone (a corticosteroid drug), which, although it does not cure, it slows down the decrease in muscle strength and delays the onset of disability. It is believed that corticosteroids, such as prednisone, act by blocking the activation of immune cells and infiltration, which are accelerated by damage to muscle fibers resulting from the disease. Unfortunately, corticosteroid treatment also leads to skeletal muscle atrophy, which negates some of the likely benefits of blocking the immune response in these patients. Thus, there remains a need to identify therapeutic agents that slow down damage to muscle fibers and delay the onset of disability in patients with muscular dystrophy, but to a lesser extent cause skeletal muscle atrophy than modern treatments.

SUMMARY OF THE INVENTION

The present invention provides isolated peptides that are CRF ₂ R agonists. In particular, the invention provides an isolated peptide or nucleic acid encoding it, which are derivatives of CRF, urocortin I, urocortin II, urocortin III, sauvagin, urotensin I or related peptides. The invention also relates to a pharmaceutical composition comprising a safe and effective amount of an isolated peptide according to this invention and a pharmaceutically acceptable excipient. The invention also relates to a kit containing an isolated peptide in a dosage form and instructions for use.

The administration of a peptide or nucleic acid encoding a peptide, a pharmaceutical composition or kit according to this invention to a subject in need thereof is effective for treating modulated CRF ₂ R disorders, such as atrophy or muscle wasting. The invention also relates to an antibody that is specific for the peptides of this invention. Finally, the invention relates to the use of a peptide of the invention or a nucleic acid encoding it for the manufacture of a medicament for the treatment of a modulated CRF ₂ R disorder in a subject in need thereof.

In one of the embodiments the invention relates to the amino acid sequence:

IVLSLDVPIGLLQILLEQX ₁₉ KX ₂₁ X ₂₂ X ₂₃ X ₂₄ X ₂₅ QATTNARILARV (SEQ ID NO: 531)

Where:

(a) X _{19 is} selected from the group consisting of D and E;

(b) X _{21 is} selected from the group consisting of A and Q;

(c) X _{22 is} selected from the group consisting of R, E, and K;

(d) X _{23 is} selected from the group consisting of A, K, and N;

(e) X _{24 is} selected from the group consisting of A, E, and L;

(f) X _{25 is} selected from the group consisting of R and K; and

(g) X _{26 is} selected from the group consisting of E and Q.

The present invention encompasses isolated non-native peptides according to formula (I):

Where:

(a) alpha contains a sequence of the formula X ₁ X ₂ X ₃ X ₄ X ₅ X ₆ , where:

each of X ₁ , X ₂ and X _{3 is} selected from the group consisting of zero. A, E, D, G, N, P, Q, S, T, and Z;

X _{4 is} selected from the group consisting of F, I, L, P, T, and V;

X _{5 is} selected from the group consisting of A, I, P, S, T, and V;

X _{6 is} selected from the group consisting of I, L, M, and N;

(b) beta contains the sequence of the formula SX ₈ DX ₁₀ ; where: each of X ₈ and X _{10 is} independently selected from the group consisting of I, L and V;

(c) the gamma contains the sequence of the formula X ₁₁ X ₁₂ X ₁₃ ; where: X _{11 is} selected from the group consisting of P, T, V and S, and each of X ₁₂ and X _{13 is} independently selected from the group consisting of A, naphthylalanine (depicted as B), C, D, E, F , G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y;

(d) the delta contains a sequence of the formula X ₁₄ X ₁₅ X ₁₆ , where:

X _{14 is} selected from the group consisting of I, L, and M;

X _{15 is} selected from the group consisting of L and M; and

X _{16 is} selected from the group consisting of S, N, Q, and R;

(e) epsilon contains a sequence of the formula X ₁₇ X ₁₈ X ₁₉ X ₂₀ X ₂₁ , where:

X _{17 is} selected from the group consisting of V, I, L, T, K, E, N, and Q;

X _{18 is} selected from the group consisting of L, M, V, A, and T;

X _{19 is} selected from the group consisting of I, F, L, and M;

X _{20 is} selected from the group consisting of D, E, N, and H; and

X _{21 is} selected from the group consisting of L, V, I, Q, M, and R;

(f) the zeta contains a sequence of the formula X ₂₂ X ₂₃ X ₂₄ X ₂₅ , where:

X _{22 is} selected from the group consisting of zero, A, D, E, S, and T;

X _{23 is} selected from the group consisting of zero, K and R;

X _{24 is} selected from the group consisting of zero, A, H, M, N, Q, T, and Y;

X _{25 is} selected from the group consisting of zero, E, D, I, K, N, Q and R;

(g) this contains a sequence of the formula X ₂₆ X ₂₇ X ₂₈ X ₂₉ X ₃₀ X ₃₁ , where:

X _{26 is} selected from the group consisting of A, D, G, H, K, N, Q, and S;

X _{27 is} selected from the group consisting of A, E, I, L, M, and Q;

X _{28 is} selected from the group consisting of A, H, K, Q, R, and V;

X _{29 is} selected from the group consisting of A, E, K, N, M, and Q;

X _{30 is} selected from the group consisting of H, K, N, Q, and R;

X _{31 is} selected from the group consisting of A and K;

(h) theta contains the sequence of the formula X ₃₂ X ₃₃ NX ₃₅ X ₃₆ X ₃₇ X ₃₈ X ₃₉ X ₄₀ X ₄₁ , where:

X _{32 is} selected from the group consisting of A, E, H, and T;

X _{33 is} selected from the group consisting of A, D, E, I, L, N, Q, R, S, and T;

X _{35 is} selected from the group consisting of A and R;

X _{36 is} selected from the group consisting of E, H, I, K, L, N, Q, and R;

X _{37 is} selected from the group consisting of F, I, L, M, and Y;

X _{38 is} selected from the group consisting of L, F, and M;

X _{39 is} selected from the group consisting of A, D, E, N, and Q;

X _{40 is} selected from the group consisting of A, D, E, H, I, K, N, Q, R, S, and T;

X _{41 is} selected from the group consisting of A, F, I, and V; and their options.

All cited documents in the relevant part are incorporated into this description by reference; citation of any document should not be construed as recognition that it represents the prior art in relation to this invention.

DESCRIPTION OF THE LIST OF SEQUENCES

Table 1 describes the various sequences of proteins and protein fragments that bind to CRF receptors. These selected sequences are included with the corresponding access number (s) in Genbank or Derwent and the name of the species of animal for which they are published, as well as access numbers for related nucleotide sequences that encode identical or almost identical amino acid sequences. These known and new sequences according to the invention, in addition, are presented in the list of sequences.

Table 1 Description
sequences Amino Acid SEQ ID NO: View Access number for the nucleotide sequence in Genbank (GB), Swiss-Prot (SP) or Derwent (D) Associated access numbers in Genbank (GB) or Derwent (D); fragment of urocortin I 2 Homo sapiens Fragment AF038633 (GB) amino acid residues 83-122 AC109828 (GB)
AX015619 (GB)
AV708591 (GB)
AV708591 (GB)
AAZ35707 (D)
AAT73432 (D) fragment of urocortin II four Homo sapiens Fragment AF320560 (GB) amino acid residues 72-109 fragment of urocortin III 6 Homo sapiens Fragment AF361943 (GB) amino acid residues 118-157 AY026949 (GB) hormone corticotropin releasing fragment 8 Homo sapiens Fragment V00571 (GB) amino acid residues 154-194 AC090195 (GB)
AC090196 (GB)
BC002599 (GB)
AC021240 (GB)
E00245 (GB) fragment of corticotropin releasing factor 10 Ovis. sp. E00212 (GB) J00803 (GB)
M22853 (GB) sauvagin eleven Phyllomedusa sauvagei P01144 (SP)

DESCRIPTION OF THE INVENTION

Glossary of Terms

The following is a list of definitions of terms used in this description.

"Agonist" means any compound, including, but not limited to, an antibody that activates a receptor. For example, CRFR agonists include, but are not limited to, CRF, urocortin, urocortin II, urocortin III, urotensin I, sauvagin, and related analogues.

“Antibody” in its various grammatical forms means immunoglobulin molecules and immunologically active parts of immunoglobulin molecules, i.e. molecules that contain an antigen binding site that specifically binds antigen. As used herein, an “isolated antibody” means an antibody that has been partially or completely separated from proteins and naturally occurring organic molecules with which it is associated in nature.

“Binding affinity” means the ligand's tendency to interact with the receptor and is inversely related to the dissociation constant of the specific CRF ligand interaction — CRFR. The dissociation constant can be directly measured using standard methods of saturation, competitive binding or determination of binding kinetics, or indirectly using pharmacological methods that include functional assays and end results.

"Chimeric antibody" means an antibody that contains structural elements from two or more different antibody molecules, i.e. from different types of animals. Chimeric antibodies include, but are not limited to, antibodies known as “humanized antibodies”, which include, but are not limited to, chimeric antibodies formed by a method known as grafting complementarity determining regions.

“CRF” means corticotropin releasing hormone, which means the same as corticotropin releasing hormone (CRH). Examples of CRF peptides include r / h-CRF and sheep CRF (see US Pat. No. 4,415,558) and the like.

“CRF analogue” means substances that act as CRFR ligands. Suitable CRF analogs can be obtained from various types of vertebrates, and they include, but are not limited to, substances such as sauwagin (see, for example, US Pat. No. 4,605,642), urotensin (see, for example, US Pat. No. 4,908,352 and 4533654), mouse urocortin II, human urocortin-related peptide (Reyes, TM et al., Proc. Natl. Acad. Sci. 98: 2843-2848 (2001)), urocortin (see, for example, WO 97/00063), human urocortin II (peptide related to streskopin), human urocortin III (streskopin), dog fish URP 1, dog fish URP II, urotensin I and CRF analogues described in US Pat. Nos. 4,415,558; 4,489,163; 4,594,329; 4,605,642; 5,109,111; 5,235,036; 5,278,146; 5,439,885; 5,493,006; 5,663,292; 5,824,771; 5844074 and 5869450. Specific CRF analogues include hUcnI (human urocortin I, AF038633 (GB)); hUroII (human urocortin II or peptide related to streskopin) (AF320560); hUroIII (human urocortin III or streskopin, AF361943); hCRF (human corticotropin releasing factor) (V00571 (GB)); oCRF (corticotropin releasing sheep factor E00212 (GB)); Svg (Sauvagin, P01144 (SP)).

"CRFR agonist" means a compound or molecule that has the ability to activate CRF ₁ R, CRF ₂ R, or both receptors.

“CRFR” means CRF ₁ R or CRF ₂ R. The term “CRFR” also includes truncated and / or mutant proteins in which regions of a receptor molecule not required for ligand binding or signal transmission have been deleted or modified.

"CRF ₁ R" means any isoform of CRF ₁ R from any animal species. CRF ₁ R was previously called CRF-RA, PC-CRF, CRF, (Perrin, MH, et al. Endocrinology 133: 3058-3061 (1993), Chen, R., et al. Proc. Natl. Acad. Sci. USA 90: 8967-8971 (1993), Chang, CP. Et al., Neuron 11: 1187-1195 (1993), Kishimoto, T., et al., Proc. Natl. Acad. Sci. USA, 92: 1108- 1112 (1995) and Vita, N., et al., FEBS Lett. 335: 1-5 (1993)) or CRH receptor.

The definition of CRF ₁ R includes, but is not limited to, those receptors for which a cDNA or genomic sequence encoding a receptor has been deposited in a sequence database. The indicated sequences include sequences with access numbers: X72304, E11431, L23332, I92584, T37068, T28968, Q81952, L23333, NM_004382, AF180301, T28970, L25438, L24096, I92586, Q81954, AH006791, NM_30962, AF459240, AF40540140540 AF229361, AB055434 and L41563. The nucleotide and protein sequences of these receptors are available from GenBank or Derwent.

"CRF ₂ R" means any isoform of CRF ₂ R from any animal species. CRF ₂ R is also called HM-CRF, CRF-RB, (Kishimoto, T., et al., Proc. Natl. Acad. Sci. USA, 92: 1108-1112 (1995) and Perrin, M., et al. Proc. Natl. Acad. Sci. USA 92: 2969-2973 (1995)).

The definition of a CRF ₂ R receptor includes, but is not limited to, those receptors for which the DNA sequence encoding the receptor has been deposited in a sequence database. These sequences include sequences with access numbers: U34587, E12752, NM_001883, T12247, T66508, AF011406, AF019381, U16253, T12244, T28972, U17858, NM_009953, Y14037 and AF229360. The nucleotide and protein sequences of these receptors are available from GenBank or Derwent.

"Inhibit" means the partial or complete blocking of a particular process or activity. For example, a compound inhibits skeletal muscle atrophy if it either completely or partially prevents muscle atrophy.

“Isolated peptide” means that the peptide molecule is said to be “isolated” when physical, mechanical, or chemical methods are used to extract the peptide from cellular components that are usually associated with a protein. One of skill in the art can easily use standard purification methods to produce an isolated peptide.

"Isolated nucleic acid" means that the nucleic acid molecule is essentially separated from contaminating nucleic acid molecules encoding other polypeptides. Methods for purification and sequence identification are well known in the art.

In the sense used in this description, it is said that two DNA sequences are “functionally linked” if the nature of the link between the two DNA sequences (1) is not the result of introducing a frameshift mutation, (2) does not interfere with the ability of the promoter region to control transcription of coding sequences or (3) does not interfere with the ability of the corresponding RNA transcript to translate into protein. For example, the coding sequence and regulatory sequences are operably linked when they are covalently linked in such a way as to place transcription of the coding sequence under the influence or control of regulatory sequences. Thus, the promoter region is functionally linked to the coding sequence when the promoter region is capable of transcribing a given DNA sequence so that the resulting transcript has the ability to translate into the desired peptide.

"Selective agonist" means that the agonist, as a rule, has a greater, preferably significantly greater activity in relation to a particular receptor (s) compared to other receptors, and not that it is completely inactive in relation to other receptors.

"Sequence identity" or "homology" at the level of amino acid or nucleotide sequences is determined using the BLAST analysis (Basic Local Alignment Search Tool), using the algorithm used in the programs blastp, blastn, blastx, tblastn and tblastx (Altschul et al. (1997) Nucleic Acids Res. 25, 3389-3402 and Karlin et al. (1990) Proc. Natl. Acad. Sci. USA 87, 2264-2268), which are specifically designed to search for sequence similarity. The approach used in the BLAST program is to first consider similar sections with spaces (intermittent) and without spaces (continuous) between the requested sequence and the sequence in the database, then evaluate the statistical significance of all matches that are identified, and finally summarize only those matches that satisfy the pre-selected threshold of significance. For a discussion of the main problems when looking for similarities in a sequence database, see Altschul et al. (1994) Nature Genetics 6, 119-129. The search parameters for the histogram, descriptions, alignments, expectations (i.e. the threshold of statistical significance for indicated matches with sequences in the database), clipping, matrix, and filter (low complexity) are the default parameters. The default scoring matrix used in blastp, blastx, tblastn and tblastx is the BLOSUM62 matrix (Henikoff et al. (1992) Proc. Natl. Acad. Sci. USA 89, 10915-10919), recommended for requested sequences above 85 nucleotides or amino acids in length.

In the case of blastn, the score matrix is set based on the ratio of M (i.e. the number of reward points for a pair of matching balances) and N (i.e. the number of penalty points for mismatched balances), where the default values of M and N are +5 and -4, respectively. Four blastn parameters were adjusted as follows: Q = 10 (penalty for gap formation); R = 10 (penalty for widening the gap); wink = 1 (generates word hits in each position currently being viewed along the query) and gapw = 16 (sets the width of the window in which alignments with spaces are created). The equivalent Blastp parameter settings were Q = 9; R is 2; wink = 1 and gapw = 32. The Bestfit comparison between sequences, available in GCG, version 10.0, uses the options for DNA GAP = 50 (fine for creating a gap) and LEN = 3 (fine for widening a gap), and the equivalent settings for protein comparisons are GAP = 8 and LEN = 2.

“Skeletal muscle hypertrophy” means an increase in skeletal muscle mass, or skeletal muscle function, or both.

“Skeletal muscle atrophy” means the same as “muscle wasting”, and means a decrease in skeletal muscle mass, or skeletal muscle function, or both.

When describing the structure and function of proteins, indications are given of the amino acids that make up the protein. Amino acids can also be indicated by their usual abbreviations, as shown: A = Ala = alanine; T = Thr = threonine; V = Val = valine; C = Cys = cysteine; L = Leu = Leucine; Y = Tyr = tyrosine; I = Ile = isoleucine; N = Asn = Asparagine; P = Pro = proline; Q = Gln = Glutamine; F = Phe = phenylalanine; D = Asp = aspartic acid; W = Trp = tryptophan; E = Glu = glutamic acid; M = Met = methionine; K = Lys = lysine; G = Gly = glycine; R = Arg = arginine; S = Ser = Serine; H = His = histidine. The letter Z = Glx = pyrrolidone carboxylic acid is used to denote the N-terminal glutamic acid or glutamine that formed the inner cyclic lactam. This is described in the list of sequences in the form of a sign of “modified residue”, where appropriate. The letter B is used in the description to denote naphthylalanine, a modification of alanine in some peptides, and this is indicated in the list of sequences as “another sign” in the list of sequences in the peptide sequences where this occurs. The abbreviation "Ac" is used to denote the modified acetylated NH ₂ end in the description and is described as the "modified residue" sign, where appropriate. The peptides of the invention are also modified to have an amide group at the carboxyl end. This is indicated in the list of sequences in the form of a characteristic “modified residue”. To indicate a deletion or absence of an amino acid in the context of a natural homologue, “-” or “zero” is used throughout the application.

Unless otherwise specified, all technical and scientific terms used in this description have the same meaning as the meaning usually implied by a specialist in the field of protein chemistry, pharmacology or molecular biology. The methods, substances and examples described in this application are not intended to be limiting. Other methods and substances similar or equivalent to the methods and substances described in this application can be used in practice or in testing this invention.

Peptides

The present invention encompasses isolated non-native peptides according to formula (I):

alpha beta gamma delta epsilon zeta eta theta

(I)

In formula (I), alpha contains a sequence of the formula X ₁ X ₂ X ₃ X ₄ X ₅ X ₆ , where: each of X ₁ , X ₂ and X _{3 is} selected from the group consisting of zero, A, E, D, G, N, P, Q, S, T, and Z; X _{4 is} selected from the group consisting of F, I, L, P, T, and V; X _{5 is} selected from the group consisting of A, I, P, S, T, and V; and X _{6 is} selected from the group consisting of I, L, M, and N. In one aspect of the invention, alpha contains a sequence of the formula X ₁ X ₂ X ₃ X ₄ X ₅ X ₆ , where X ₁ is zero, X _{2 is} selected from the group, consisting of D, E and Z; X _{3 is} selected from the group consisting of D, G, and N; X _{4 is} selected from the group consisting of L and P; X _{5 is} selected from the group consisting of P and S; and X _{6 is} selected from the group consisting of I, L, M, and N. In one embodiment, alpha also contains a sequence selected from the group consisting of -EDLPL (SEQ ID NO: 388), -DNPSL (SEQ ID NO : 389), -DDPPL (SEQ ID NO: 390), -ZGPPI (SEQ ID NO: 391), --- PSL and --- IVL, where “-” means zero. In another embodiment, the alpha contains the sequence -ZGPPI. In another embodiment, the alpha contains the sequence -DNPSL. In another embodiment, the alpha contains the sequence --- IVL. In another embodiment, the alpha contains the sequence --- PSL.

In another aspect of the invention, alpha comprises a sequence of the formula X ₁ X ₂ X ₃ X ₄ X ₅ X ₆ , where X ₁ is zero; X ₂ is zero; X ₃ is zero; X _{4 is} selected from the group consisting of F, I, L, P, and V; X _{5 is} selected from the group consisting of A, I, S, T, and V; X ₆ is L. In one embodiment, the alpha contains a sequence selected from the group consisting of --- IVL, --- FTL, --- LTL, --- FAL, --- VIL and --- PSL. In another embodiment, the alpha contains the sequence --- IVL.

In another aspect of the invention, alpha contains a sequence selected from the group consisting of SQEPPI (SEQ ID NO: 392), SEEPPI (SEQ ID NO: 393), -DNPSL, --- IVL, -TKFTL (SEQ ID NO: 394) , -ZGPPI, SQEIVL (SEQ ID NO: 395), SEEIVL (SEQ ID NO: 396), DNPIVL (SEQ ID NO: 397), TKIVL (SEQ ID NO: 398), ZGIVL (SEQ ID NO: 399), SDNPSL (SEQ ID NO: 401), STKFTL (SEQ ID NO: 402), SZGPPI (SEQ ID NO: 403), and NDDPPI (SEQ ID NO: 404).

In yet another aspect of the invention, alpha may be preceded by polyhistidine (HHHHHH, SEQ ID NO: 400) or another peptide label that can be used in the purification or detection of peptides according to the invention.

In formula (I), beta contains a sequence of formula SX ₈ DX ₁₀ , where each X ₈ and X _{10 is} selected from the group consisting of I, L and V. In one embodiment, beta contains a sequence selected from the group consisting of SIDL (SEQ ID NO: 405), SLDV (SEQ ID NO: 406), SLDL (SEQ ID NO: 407), SIDI (SEQ ID NO: 408) and SIDV (SEQ ID NO: 409). In another embodiment, the beta contains a sequence, in addition, selected from the group consisting of SIDL and SLDV. In yet another embodiment, the beta contains the SIDL sequence. In yet another embodiment, the beta contains the sequence of SLDV. In yet another embodiment, the beta contains the SIDV sequence.

In formula (I), the gamma contains the sequence of the formula X ₁₁ X ₁₂ X ₁₃ ; where X ₁₁ means PT, V or S and each X ₁₂ and X _{13 is} selected from the group consisting of A, B (naphthylalanine), C, D, E, F, G, H, I, K, L, M, N , P, Q, R, S, T, V, W, and Y. In one embodiment, X ₁₁ is P. In another embodiment, the gamma contains a sequence selected from the group consisting of PAB, PAF, PAH, PAQ, PAY, PFB , PFE, PFF, PFG, PFH, PFI, PFL, PFQ, PFV, PFW, PFY, PGY, PHB, PHF, PHH, PHQ, PHW, PHY, PIA, PIB, PID, PIE, PIF, PIG, PIH, PII , PIL, PIQ, PIR, PIT, PIV, PIW, PIY, PKY, PLB, PLE, PLF, PLG, PLH, PLI, PLL, PLQ, PLV, PLW, PLY, PNY, PQB, PQF, PQH, PQI, PQL , PQQ, PQV, PQW, PQY, PRY, PSY, PTB, PTE, PTF, PTH, PTI, PTL, PTV, PTW, PTY, PVB, PVY, PWF, PWH, PWQ, PWW, PWY, PYB, PYF, PYH , PYI, PYL, PYQ, PYT, PYV, PYW, PYY, SLE, SLG, SIG, and VIG. In yet another embodiment of the invention, the gamma contains a sequence selected from the group consisting of PFE, PFG, PFH, PFQ, PFY, PLE, PLG, PLH, PLQ, PLY, PTE, PTH, PTY, PIE, PIH, PIQ, PIY, PIG , PTN, and PTS. In yet another embodiment, the gamma contains a sequence selected from the group consisting of PFE, PFG, PFH, PFQ, PFY, PLE, PLG, PLH, PLQ, PLY, PTE, PTH, PTY, PIE, PIH, PIQ, PIY, PYY, PFE, PTW, PQY, PHY, PII, PIL, PTI, PTF, PTL, PIV, PIT, PTV and PIE. In yet another embodiment, the gamma contains a sequence selected from the group consisting of PIG, PTN, PTS and PIG. In yet another embodiment, the gamma contains a sequence selected from PFQ, PYW, PLQ, PIG, PLY, PUY, PTY, PIG, PLL, PLF and PFF. In yet another embodiment, the gamma contains the PIG sequence. In yet another embodiment, the gamma contains PFQ.

In formula (I), the delta contains a sequence of the formula X ₁₄ X ₁₅ X ₁₆ , where X _{14 is} selected from the group consisting of I, L and M; X _{15 is} selected from the group consisting of L and M; and X _{16 is} selected from the group consisting of S, N, Q, and R. In one embodiment, the delta contains a sequence selected from the group consisting of ILS, IMN, LLQ, LLR, and MLR. In one embodiment, the delta contains an LLQ or LLR sequence.

In formula (I), epsilon contains a sequence of the formula X ₁₇ X ₁₈ X ₁₉ X ₂₀ X ₂₁ , where X _{17 is} selected from the group consisting of V, I, L, T, K, E, N and Q; X _{18 is} selected from the group consisting of L, M, V, A, and T; X _{19 is} selected from the group consisting of I, F, L, and M; X _{20 is} selected from the group consisting of D, E, N, and H; and X _{21 is} selected from the group consisting of L, V, I, Q, M and R. In one embodiment, the epsilon comprises a sequence selected from the group consisting of VLIDL (SEQ ID NO: 410), VLFDV (SEQ ID NO: 411 ), VLIEI (SEQ ID NO: 412), ILFNI (SEQ ID NO: 413), LLIEI (SEQ ID NO: 414), LLFNI (SEQ ID NO: 415), ILLEQ (SEQ ID NO: 416), ILIEI (SEQ ID NO: 417), ILLEI (SEQ ID NO: 418), TLLEL (SEQ ID NO: 419), KMIEI (SEQ ID NO: 420), KVIEI (SEQ ID NO: 421), EVLEM (SEQ ID NO: 422) , EMIEI (SEQ ID NO: 423), EVIEI (SEQ ID NO: 424), EAIEI (SEQ ID NO: 425), ETIEI (SEQ ID NO: 426), EIIEI (SEQ ID NO: 427), ELIEI (SEQ GO NO: 428), NMIEM (SEQ ID NO: 429), NMIHR (SEQ ID NO: 430), NMIHM (SEQ ID NO: 431), QMMEM (SEQ ID NO: 432), and LLFNI (SEQ ID NO: 433) . In one embodiment of the invention, the epsilon comprises a sequence selected from the group consisting of VLIDL, VLFDV, ILFNI, LLFNI, ILLEQ, TLLEL and KMIEI. In another embodiment, the epsilon comprises a sequence selected from the group consisting of VLIDL, VLFDV, ILFNI, and ILLEQ. In yet another embodiment, the epsilon comprises a sequence selected from the group consisting of KMIEI or ILLEQ. In yet another embodiment, the epsilon comprises the sequence KVIEI, KMIEI, ILLEI, ILLEQ or TLLEL. In yet another embodiment, the epsilon comprises a KMIEI sequence. In yet another embodiment, the epsilon comprises an ILLEQ sequence.

In formula (I), the zeta contains a sequence of the formula X ₂₂ X ₂₃ X ₂₄ X ₂₅ , where X _{22 is} selected from the group consisting of zero, A, D, E, S, and T; X _{23 is} selected from the group consisting of zero, K and R; X _{24 is} selected from the group consisting of zero, AH, M, N, Q, T, and Y; and X _{25 is} selected from the group consisting of zero, E, D, I, K, N, Q, and R. In one embodiment of the invention, the zeta contains a sequence of the formula X ₂₂ X ₂₃ X ₂₄ X ₂₅ ; where X _{22 is} selected from the group consisting of zero, D and E; X _{23 is} selected from the group consisting of zero, K and R; X _{24 is} selected from the group consisting of zero, A, H, M, N, Q, T, and Y; X _{25 is} selected from the group consisting of zero, E, D, I, K, N, Q, and R. In another embodiment, the zeta contains a sequence selected from the group consisting of SRAE (SEQ ID NO: 434), EKAR (SEQ ID NO: 435), ERAR (SEQ ID NO: 436), EKQE (SEQ ID NO: 437), TKDR (SEQ ID NO: 438), TKAD (SEQ ID NO: 439), AKAR (SEQ ID NO: 440), AKQR (SEQ ID NO: 441), ERQR (SEQ ID NO: 442), AKAE (SEQ ID NO: 443), ERAE (SEQ ID NO: 444), ARQR (SEQ ID NO: 445), EKQR (SEQ ID NO : 446), TKAN (SEQ ID NO: 447), TKAR (SEQ ID NO: 448), EAAR (SEQ ID NO: 449), ERQE (SEQ ID NO: 450), ARAD (SEQ ID NO: 451), EKTQ (SEQ ID NO: 452), ARAR (SEQ ID NO: 453), ARAE (SEQ ID NO: 454), ARQE (SEQ ID NO: 455), AKQE (SEQ ID NO: 456), TRAD (SEQ ID NO: 457), AKAD (SEQ ID NO: 458), TRAR (SEQ ID NO: 459), EKQQ (SEQ ID NO: 520), --RR, --AA, -AAR, --- R, -RAR, - --A, --AR, -ARA, -RR, A-AR, AA-, A ---, ARA- and ----. In yet another embodiment, the zeta contains a sequence selected from the group consisting of EKAR, ERAR, EKQE and TKDR. In yet another embodiment, the zeta contains EKQE, EKTQ, ARAR, or EKAR.

In formula (I), this contains the sequence of the formula X ₂₆ X ₂₇ X ₂₈ X ₂₉ X ₃₀ X ₃₁ , where: X _{26 is} selected from the group consisting of A, D, G, H, K, N, Q and S; X _{27 is} selected from the group consisting of A, E, I, L, M, and Q; X _{28 is} selected from the group consisting of A, H, K, Q, R, and V; X _{29 is} selected from the group consisting of A, E, K, M, N, and Q; X _{30 is} selected from the group consisting of H, K, N, Q, and R; and X _{31 is} selected from the group consisting of A and K. In one embodiment of the invention, this contains a sequence selected from the group consisting of AAREQA (SEQ ID NO: 460); KEKKRK (SEQ ID NO: 461); SQRERA (SEQ ID NO: 462), KEKQQA (SEQ ID NO: 463), and QLAQQA (SEQ ID NO: 464) AARNQA (SEQ ID NO: 521), KERNQA (SEQ ID NO: 522), KEKNQA (SEQ ID NO : 523), KQRERA (SEQ ID NO: 524), KERERA (SEQ ID NO: 525), KEKERA (SEQ ID NO: 526), KEKQRA (SEQ ID NO: 527), AEAAAK (SEQ ID NO: 528), AAHAAA (SEQ ID NO: 529), and HAHAHA (SEQ ID NO: 530). In yet another embodiment, this contains a sequence selected from the group consisting of AAREQA and KEKKRK. In yet another embodiment, this contains the sequence AAREQA. In yet another embodiment, this contains a sequence selected from the group consisting of SQRERA and KEKQQA. In yet another embodiment, this contains the sequence KEKQQA.

In formula (I), the theta contains a sequence of the formula X ₃₂ X ₃₃ N ₃₄ X ₃₅ X ₃₆ X ₃₇ X ₃₈ X ₃₉ X ₄₀ X ₄₁ , where X _{32 is} selected from the group consisting of A, E, H and T; X _{33 is} selected from the group consisting of A, D, E, I, L, N, Q, R, S, and T; X _{35 is} selected from the group consisting of A and R; X _{36 is} selected from the group consisting of E, H, I, K, L, N, Q, and R; X _{37 is} selected from the group consisting of F, I, L, M, and Y; X _{38 is} selected from the group consisting of L, F, and M; X _{39 is} selected from the group consisting of A, D, E, N, and Q; X _{40 is} selected from the group consisting of A, D, E, H, I, K, N, Q, R, S, and T; X _{41 is} selected from the group consisting of A, F, I and V. In one embodiment of the invention, the theta contains a sequence of the formula X ₃₂ X ₃₃ NX ₃₅ X ₃₆ X ₃₇ X ₃₈ X ₃₉ X ₄₀ X ₄₁ , where X _{32 is} selected from the group consisting of A, E and T; X _{33 is} selected from the group consisting of A, D, E, N, Q, S, and T; X _{35 is} selected from the group consisting of A and R; X _{36 is} selected from the group consisting of H, I, L, N, Q, and R; X _{37 is} selected from the group consisting of F, I, L, M, and Y; X _{38 is} selected from the group consisting of L, F, and M; X _{39 is} selected from the group consisting of A, D, E, N, and Q; X _{40 is} selected from the group consisting of zero, A, D, H, Q, R, S, and T; X _{41 is} selected from the group consisting of I and V. In another embodiment, the theta contains a sequence selected from the group consisting of AANRLLLDTV (SEQ ID NO: 465), AAQEQILAHV (SEQ ID NO: 466), ANNAELLAEI (SEQ ID NO: 467 ), ANNAHLLAHI (SEQ ID NO: 468), ANNAKLLAKI (SEQ ID NO: 469), ANNALLLATI (SEQ ID NO: 470), ANNALLLDTI (SEQ ID NO: 471), ANNANLLANI (SEQ ID NO: 472), ANNAQLLAHI (SEQ ID NO: 473), ANNAQLLAQI (SEQ ID NO: 474), ANNARILARV (SEQ ID NO: 475), ANNARLLARI (SEQ ID NO: 476), ANNARLLDTI (SEQ ID NO: 477), ANNRLLLATI (SEQ ID NO: 478) , ANNRLLLDTI (SEQ ID NO: 479), EQNAHIFAHV (SEQ ID NO: 480), EQNAQIFAHV (SEQ ID NO: 481), EQNARIFARV (SEQ ID NO: 482), EQNRIIFDSV (SEQ ID NO: 483), ETNARILARV (SEQ ID NO: 483) NO: 484), HAQAHILAHV (SEQ ID NO: 485), HSNRKIIDIA (SEQ ID NO: 486), HSNRKLLDIA (SEQ ID NO: 487), HSNRKLMEII (SEQ ID NO: 488), HTNARILARV (SEQ ID NO: 489), TNNRLLLATV (SEQ ID NO: 490), TNNRLLLDTI (SEQ ID NO: 491), TSNRKLMEII (SEQ ID NO: 492), TTNARILARN (SEQ ID NO: 493), TTNARILARV (SEQ ID NO: 494), TTNARLLATV (SEQ ID NO: 495), TTNARLLDRV (SEQ ID NO: 496), TTNARLLDTV (SEQ ID NO: 497), TTNRLLLARV (SEQ ID NO: 498), TTNRLLLATV (SEQ ID NO: 499), TTNRLLLDTV (SEQ ID NO: 500), TTQARILARV (SEQ ID NO: 501) and TTVARILARV (SEQ ID NO : 502). In yet another embodiment, the theta contains a sequence selected from the group consisting of TTNARILARV, ANNALLLDTI, ANNALLLATI, TTNARLLDTV and TTNARLLDRV. In yet another embodiment, the theta contains the sequence ANNARLLDTI, ANNARLLARI, ANNALLLDTI, ANNALLLATI, TTNARLLDRV, TTNARILARV, ANNRLLLDTI, EQNARIFARV, EQNAHIFAHV and EQNAQIFAHV. One skilled in the art will readily understand that theta includes the C-terminus of a peptide.

The peptides of the invention have also been described as peptides of 41 amino acids with specific preferred sequences. The following peptide chains are given as specific examples: ZGPPISIDLP (SEQ ID NO: 503) for residues X ₂ -X ₁₁ , LLRK (SEQ ID NO: 504) for residues X ₁₄ -X ₁₇ , IEIEKQEKEKQQA (SEQ ID NO: 505) for residues X ₁₉ -X ₃₁ , PSLSID (SEQ ID NO: 506) for residues X ₄ -X ₉ and LLRTLLELEKTQSQRERAEQNA (SEQ ID NO: 507) for residues X ₁₄ - ₃₅ .

Variants of the described peptides and their nucleotide sequences encoding them are also included in the scope of this invention. As used herein, the term “variants” means those peptides, polypeptides or proteins or nucleotide sequences encoding them that are substantially similar to the peptides described by formula (I), and which can be used as CRF ₂ R agonists. A peptide of formula ( I) can be modified in various ways to obtain a variant of the peptides included in the scope of this invention, including amino acid substitutions, deletions, truncations, insertions and modifications. Methods of such manipulations are widely known in the art. For example, variants can be obtained using mutations in the nucleotide sequences encoding them. Methods for mutagenesis and changes in nucleotide sequences are well known in the art. See, for example, Kunkel (1985) Proc. Natl. Acad Sci. USA 82: 488-492; Kunkel et al. (1987) Methods in Enzymol. 154: 367-382; U.S. Patent No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York) and references cited in these publications. In one case of the variant, the replacement (s) in the peptide of formula (I) is conservative, since it minimally violates the biochemical properties of the variant. Thus, when mutations are introduced to replace amino acid residues, positively charged residues (H, K and R) are preferably replaced by positively charged residues; negatively charged residues (D and E) are preferably replaced by negatively charged residues; and neutral non-polar residues (A, F, I, L, M, P, V and W) are preferably replaced with neutral non-polar residues. In another case of the variant, the total charge, structure or hydrophobic / hydrophilic properties of the peptide can be changed without significant adverse effect on the agonism of CRF ₂ R. In another case, the variant is the active fragment of the peptide of formula (I). In yet another embodiment, the peptides of formula (I) are modified by acetylation, carboxylation, phosphorylation, glycosylation, ubiquitination, and labeling by enzymatic processing of the protein either in vivo or in vitro or by synthesis of the peptide using modified amino acids. Common non-limiting examples of amino acid modifications include phosphorylation of tyrosine, serine and threonine residues; methylation of the lysine residue; acetylation of lysine residues; hydroxylation of proline and lysine residues; carboxylation of glutamic acid residues; glycosylation of serine, threonine or asparagine residues; and ubiquitination of lysine residues. The variant may also contain other domains, such as epitope tags and His tags (for example, the peptide may be a fusion protein).

In another case, the concept of variant includes peptide mimetics of the peptide of the formula (I). As used herein, “mimetic” means an amino acid or amino acid analog that has the same or similar functional characteristics of an amino acid. Thus, for example, an arginine analog can be an arginine mimetic if the analog contains a side chain having a positive charge at physiological pH that is characteristic of the guanidinium reaction group of the arginine side chain. Examples of organic molecules that may be suitable mimetics are listed in Table 1 of US Pat. 5807819. Typically, a variant or a nucleic acid sequence encoding it according to this invention will possess at least 70%, usually 80%, preferably up to 90%, more preferably 95%, even more preferably 97%, and even more preferably 98 % and most preferably 99% sequence identity with the corresponding native amino acid sequence. It should not be considered that fusion proteins or N-terminal, C-terminal or internal extensions, deletions or insertions in the peptide sequence affect homology.

The use of the peptides according to the invention as agonists of CRF ₂ R

The peptides of the invention are useful for treating a variety of diseases, disorders and conditions that are modulated by CRF ₂ R activity or CRF ₂ R. As used herein, the terms "disease,""disorder" and "condition" are used interchangeably. As used herein, a disorder described by the terms “modulated CRF ₂ R” or “modulated by CRF ₂ R activity” refers to a disorder, condition or disease in which CRF ₂ R activity is an effective means to alleviate the disorder or one or more biological manifestations of a disease or disorder; or affects one or more points in the biological cascade, either leading to a disorder, or responsible for the underlying violation; or relieves one or more symptoms of the disorder. Thus, disorders subject to “modulation” include those disorders in which: (1) the lack of CRF ₂ R activity is the “cause” of the disorder or one or more biological manifestations, and the activity is genetically altered as a result of infection, irritation, internal stimulus or for other reasons; (2) the disease or disorder or the observed manifestation or manifestations of the disease or disorder is attenuated by the activity of CRF ₂ R (lack of CRF ₂ R activity is not necessarily causally associated with the disease or disorder or its observed manifestations); (3) CRF ₂ R activity affects a portion of a biochemical or cellular cascade that leads to a disease or disorder or is associated with a disease or disorder. In this regard, the activity of CRF ₂ R alters the cascade and therefore controls a disease, condition or disorder.

In one embodiment of the invention, the peptides according to this invention do not have or have only weak agonistic activity against CRF ₁ R. Thus, the peptides according to this invention are particularly useful for the treatment of disorders modulated by CRF ₂ R. One of such modulated CRF ₂ R disorders is skeletal muscle atrophy. Skeletal muscle atrophy can be induced by cessation of use due to surgery, bed rest, or bone fracture; nerve denervation / damage due to spinal cord injury; autoimmune disease; infectious disease; the use of glucocorticoids in the case of unrelated conditions; sepsis as a result of infection or for other reasons; limitation of nutrients due to illness or starvation; cachexia with a malignant tumor, chronic inflammation, acquired immunodeficiency syndrome (AIDS), cachexia; chronic obstructive pulmonary disease (COPD), congestive heart failure, sarcopenia and genetic disorders, such as muscular dystrophy, neurodegenerative diseases.

In another embodiment, the treatment of a modulated CRF ₂ R disorder results in an increase in skeletal muscle mass and function. Diseases and conditions affecting the mass and function of the skeletal muscle include, but are not limited to, atrophy or wasting of the skeletal muscle, including acute atrophy / wasting due to cessation of use due to illness, surgery, bed rest or accident; nerve damage due to damage to the spinal cord, autoimmune disease or infectious disease; use of glucocorticoids in case of unrelated conditions; sepsis as a result of infection or for other reasons; limitation of nutrients due to illness or starvation and space travel; and chronic atrophy / exhaustion, including cachexia in malignant tumors, chronic inflammation, cachexia associated with AIDS; COPD, congestive heart failure, genetic disorders such as muscular dystrophy, neurodegenerative diseases, and sarcopenia (age-related loss of muscle mass).

In yet another embodiment, the treatment of a modulated CRF ₂ R disorder includes the treatment of disorders affecting bones. Diseases and conditions affecting the bones include, but are not limited to, bone loss due to discontinuation of use due to illness, surgery, bed rest or accident; nerve damage due to damage to the spinal cord, autoimmune disease or infectious disease; use of glucocorticoids in case of unrelated conditions; sepsis as a result of infection or for other reasons; limitation of nutrients due to illness or starvation; and space flight. Also included is bone loss (osteoporosis) associated with age or hormones.

In another embodiment, the treatment of a modulated CRF ₂ R disorder includes the treatment of disorders affecting the heart and circulatory system, including, but not limited to, hypertension, congestive heart failure, heart damage due to a heart attack, ischemic reperfusion injury, stroke, migraine, memory loss Alzheimer's disease, dementia, etc.

In yet another embodiment, the treatment of a modulated CRF ₂ R disorder includes treating a disorder affecting the joints, including but not limited to arthritis, in particular osteoarthritis and rheumatoid arthritis.

In yet another embodiment, the treatment of a modulated CRF ₂ R disorder includes the treatment of metabolic diseases, including obesity and diabetes.

In yet another embodiment, the treatment of a modulated CRF ₂ R disorder includes: reducing pain; tumor reduction; treatment of allergic reactions, allergies; decrease in body temperature; appetite suppression; treatment of congestive heart failure; stress and anxiety; a change in undesirably low levels of secretion of adrenocorticotropic hormone ("ACTH"); control of appetite, arousal and cognitive functions; and preventing the long-term effects of stress, such as anxiety disorders, anorexia nervosa, and melancholic depression.

The term "treatment" in this description should mean at least the introduction of a peptide according to this invention, which facilitates modulated CRF ₂ R disorder in a mammalian subject, preferably a human. Thus, the term “treatment” includes: preventing a mammal from modulating CRF ₂ R disorder, especially when the mammal is predisposed to causing modulating CRF ₂ R disorder, but which has not yet been diagnosed with the disease; inhibition of modulated CRF ₂ R disorder; and / or attenuation or remission of a modulated CRF ₂ R disorder. Since the methods of this invention are directed to the prevention of a modulated CRF ₂ R disorder, it is understood that the term “prevent” does not require that the modulated CRF ₂ R disorder be completely suppressed (see Webster's Ninth Collegiate Dictionary). More precisely, in the sense used in this description, the term "prophylaxis" refers to the ability of a person skilled in the art to identify a population that is susceptible to modulated CRF ₂ R diseases so that the administration of the peptides and kits of this invention can occur before symptoms of the modulated CRF ₂ R disorder begin. A population at risk of a particular modulated CRF ₂ R disorder is easily identified. For example, a population at risk of developing muscular dystrophy can be identified by identifying mutations in the genes characteristic of the disorder. For example, the previously discussed Duchenne and Becker dystrophies result from the inheritance of a mutation in the dystrophy gene, which is located at the Xp21 locus. Those individuals in a population that have these mutations are at risk of developing muscular dystrophy. Thus, a patient population can be identified and can receive the introduction of a composition or dosage form from a kit according to this invention before the disease progresses. Thus, the progression of muscle atrophy or exhaustion in such people can be “prevented."

Nucleic acid molecules

The invention further relates to nucleic acid molecules that encode the peptides of formula (I) and their variants, preferably in isolated form. As used herein, “nucleic acid” is defined as RNA or DNA that encodes a peptide of the invention discussed above, or is a complementary nucleic acid sequence encoding such peptides. In particular, genomic DNA, cDNA, mRNA and antisense molecules are considered, as well as nucleic acids based on alternative cores or containing alternative bases obtained either from natural sources or synthesized.

The invention further relates to a fragment of a coding nucleic acid molecule. As used herein, a fragment of a coding nucleic acid molecule refers to a small portion of the entire protein coding sequence. The fragment size will be determined by the intended use. For example, if a fragment is selected to encode the active portion of a peptide according to this invention, it is necessary that the fragment is large enough to encode the functional regions of the peptide.

Fragments of encoding nucleic acid molecules of the invention (i.e., synthetic oligonucleotides) that are used as probes or specific primers for the polymerase chain reaction (PCR) or to synthesize gene sequences encoding the peptides of the invention can be easily synthesized by chemical methods, for example, the phosphotriether method of Matteucci et al., J. Am. Chem. Soc., 103: 3185-3191 (1981) or using automated synthesis methods. In addition, larger sections of DNA can easily be obtained by well-known methods, such as synthesis of a group of oligonucleotides that define different modular sections of a gene, followed by ligation of the oligonucleotides to form a complete modified gene.

The coding nucleic acid molecules of the present invention can also be modified to contain a detectable label for diagnostic and probing purposes. Many such labels are known in the art and can easily be used with the coding molecules described herein. Suitable labels include, but are not limited to, biotin, radioactively labeled nucleotides, and the like. One of skill in the art can easily use any such label to produce labeled variants of the nucleic acid molecules of the invention. Modifications of the primary structure itself by deletion, addition or alteration of amino acids included in the protein sequence during translation can be carried out without disrupting the activity of the protein. Such substitutions or other changes result in proteins having an amino acid sequence encoded by a nucleic acid that is within the scope of this invention.

Obtaining peptides or cell lines expressing peptides

The peptides according to this invention can be obtained for various applications, including, but not limited to, the use as pharmaceutical reagents for the treatment of modulated CRF ₂ R disorders. One skilled in the art will recognize that, in the case of some embodiments of the invention, purified peptides will be most applicable, while for other variants, cell lines expressing the peptides will be most applicable.

Since the peptides of formula (I) are short polypeptides, one skilled in the art will recognize that the peptides of this invention can be synthesized by direct synthesis instead of being prepared by recombinant methods using techniques well known in the art (see Bodanszky, Principles of Peptide Synthesis, Springer-Verlag, Heidelberg (1984); namely, by solid phase synthesis, see, e.g., Merrifield, J. Am. Chem. Soc., 85: 2149-54 (1963); Barany et al., Int. J. Peptide Protein Res., 30: 705-739 (1987); and U.S. Patent No. 5424398).

For example, peptides can be synthesized either using an automated synthesizer Applied Biosystem, Inc. (ABI), model 433, or using a multi-reactor synthesizer (model Symphony ^tm ) manufactured by Protein Technology, Inc. (PTI). As for the peptides synthesized using the ABI synthesizer, all reagents are purchased in ABI (with the exception of piperidine, which is purchased from Aldrich). Fmoc amino acids are purchased from ABI (except for Fmoc-L-Pyr, which is purchased from Chem-Impek). Rinka amide resins purchased from Nova Chemicals. Use standard chemistry 0.1 mmol FastMoc with a single binding. The general protocol of Fmoc chemistry for SPPS (solid-phase synthesis of peptides) includes: 1) cleavage of Fmoc-protecting groups with piperidine; 2) activation of the carboxyl group of amino acids; and 3) binding of the activated amino acids to the amino terminus of the resin bound peptide chain to form peptide bonds. Amino acids are activated with 2- (1H-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU). The dry protected amino acid in the cartridge (1.0 mmol) is dissolved in a solution of HBTU, N, N-diisopropylethylamine (DIEA) and 1-hydroxybenzotriazole (HOBt) in N, N-dimethylformamide (DMF) with the addition of additional N-methylpyrrolidone (NMP). The activated Fmoc amino acid is formed almost instantly, and the solution is transferred directly to the reaction vessel. The removal phase of the Fmoc protection is monitored and regulated by conductometric measurement. The peptide chain is built on a Rink amide resin, since a C-terminal amide is required. The final product is washed extensively with NMP and dichloromethane (DCM).

As for peptides synthesized using a multiple PTI synthesizer, all Fmoc amino acids are purchased from NovaBiochem (with the exception of Fmoc-Pyr, which is purchased from Chem-Impex). For the synthesis, the standard protocol of 0.05 mmol Fmoc synthesis is used. Fmoc amino acids (0.4 mmol) are dissolved in a solution of HBTU (200 mM), N-methylmorpholine (NMM; 0.4 M) and N, N-dimethylformamide (DMF) with the addition of N-methylpyrrolidone (NMP). The activated Fmoc amino acid is formed almost instantly, and the solution is transferred directly to the reaction vessel. The step of removing the Fmoc protection is carried out twice. The peptide chain is built on a Rink amide resin, since a C-terminal amide is required. The final synthesis product is washed extensively with NMP and dichloromethane (DCM).

Remove the protection of newly synthesized peptides. Resins containing synthesized peptides are discharged from the synthesizer and briefly dried in air. Using 1.5-2.0 ml of a cleavage mixture (containing 95% trifluoroacetic acid (TFA), 2.5% ethanodithiol, 2.5% thioanisole, 2.5% phenol (w / v) in water) in for 4 hours at room temperature, the peptides are cleaved from the resin and at the same time the side chain protecting groups [O-tert-butyl (OtBu) for Asp, Glu, Tyr, Thr and Ser are removed; pentamethylchroman-6-sulfonyl (Pmc) for Arg, tert-butoxycarbonyl (Boc) for Trp and Lys; trityl (Trt) for His, Asn and Gln] under deprotection conditions. The cleavage solution is separated from the resin by filtration. Then the filtrate is diluted with 15 ml of water. Six rounds of ether extraction are carried out to purify the peptide product. The peptide is freeze dried and stored at -20 ° C until clean.

Peptides after deprotection are purified and characterized. The peptide powder was dissolved in a 50% acetic acid solution and injected onto a Vydac C-8 column. 1.0 cm and a length of 25 cm with a particle size of 5 μm and a pore size of 300 U for cleaning. A Beckman System Gold high performance liquid chromatography (HPLC) system was used with an ultraviolet detector at two wavelengths (220 and 280 nm). A linear acetonitrile gradient is programmed and introduced onto the column to separate the peptide product from other substances. The eluate is collected using a Pharmacia fraction collector and the individual fractions after separation are subjected to both analytical HPLC (time-of-flight mass spectrometry with laser desorption and matrix ionization) and MALDI-TOF MS for characterization to ensure identity and purity.

It is also contemplated to use recombinant DNA techniques to produce peptides or cell lines expressing said peptides. Such recombinant methods are well known in the art. Methods for generating rDNA molecules are well known in the art, for example, see Sambrook et al., Molecular Cloning-A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989). In order to express recombinant peptides, according to this invention, an expression vector is obtained that contains a nucleic acid that encodes a polypeptide of interest under the control of one or more regulatory elements. The nucleic acid sequence encoding the peptides of this invention can be calculated based on the peptide sequences discussed and claimed herein.

By methods well known in the art, an isolated nucleic acid molecule encoding a peptide of interest can be ligated into a suitable expression vector. Host-expressing vector systems that can be used for the purposes of the invention include, but are not limited to: microorganisms, such as bacteria (e.g., E. coli, B. subtilis), transformed with expression vectors based on recombinant bacteriophage DNA, plasmid DNA or cosmid DNA containing nucleotide sequences encoding the peptides according to this invention; yeast (for example, Saccharomyces, Pichia) transformed with recombinant expression vectors of yeast containing nucleotide sequences encoding the peptides of this invention; insect cell systems infected with recombinant viral expression vectors (eg, baculovirus) containing nucleotide sequences encoding the peptides of this invention; plant cell systems infected with recombinant viral expression vectors (eg, cauliflower mosaic virus, tobacco mosaic virus) or transformed with recombinant plasmid expression vectors (eg, Ti plasmid) containing nucleotide sequences encoding the peptides of the invention; or mammalian cell systems (e.g., COS, CHO, HEK293, NIH3T3) carrying recombinant expression constructs containing promoters derived from the mammalian cell genome (e.g., the metallothionein promoter) or from mammalian viruses (e.g., LTR retrovirus), and also containing nucleotide sequences encoding the peptides of this invention.

In bacterial systems, a number of expression vectors can advantageously be selected depending on the intended use of the expressed peptide. For example, when a large amount of such a protein is needed, vectors that provide expression of high levels of protein products are desirable. One of skill in the art can create such vector constructs and purify proteins using a variety of techniques, including selective purification techniques, such as the use of antibody-selective or fusion protein-based columns, and non-selective purification techniques.

In insect protein expression systems, a baculovirus, A. californica nuclear polyhedrosis virus (AcNPV), is used as a vector to express foreign genes in S. frugiperda cells. In this case, the nucleotide sequences encoding the peptides of this invention are cloned in non-essential regions of the virus and placed under the control of the AcNPV promoter. Recombinant viruses are then used to infect cells in which the inserted gene is expressed, and the protein is purified by one of many methods known to one skilled in the art.

In mammalian host cells, a number of virus-based expression systems can be used. The use of these expression systems often requires the creation of specific initiation signals in vectors for efficient translation of the inserted nucleotide sequences. This is especially important when the part of the nucleotide sequence used does not contain an endogenous initiation signal. Placing the indicated initiation signal in a frame with the coding region of the inserted nucleotide sequence, as well as adding transcriptional and translational enhancer elements and purifying the recombinant protein, is carried out by one of many methods known to a person skilled in the art. Also important in the case of mammalian host cells is the selection of the appropriate type of cells that are capable of carrying out the necessary post-translational modifications of the recombinant protein. Such modifications, such as cleavage, phosphorylation, glycosylation, acetylation, etc., require the selection of an appropriate host cell that contains modifying enzymes. Such host cells include, but are not limited to, CHO, HEK293, NIH3T3, COS, etc. and known to specialists in this field.

For long-term high expression of recombinant proteins, stable expression is preferred. For example, cell lines can be constructed that stably express the peptides of this invention. One of ordinary skill in the art, following known methods such as electroporation, calcium phosphate transfection, or liposome-mediated transfection, can create a cell line that stably express the peptides of this invention. This is usually accomplished by transfection of cells using expression vectors that contain appropriate expression regulatory elements (e.g., promoter sequences, enhancer sequences, transcription termination sequences, polyadenylation sites, transcription start sites, etc.), a selectable marker and gene of interest . The selectable marker can either be in the same vector as the gene of interest, or in a separate vector that is transfected together with the vector containing the peptide encoding sequence. The selectable marker in the expression vector can confer resistance to selection and allows cells to stably integrate the vector into their chromosomes and grow, forming foci, which in turn can be cloned and propagated to obtain cell lines. Alternatively, the expression vector may allow selection of a cell expressing a selectable marker using the physical property of the marker, for example, expression of a green fluorescence protein (GFP) allows the selection of cells expressing a marker using a fluorescence activated cell sorting (FACS) assay.

One of skill in the art can select the appropriate cell type for transfection to allow selection of cells into which the sequence of interest has been successfully integrated. For example, in the case where the selectable marker is herpes simplex virus thymidine kinase, hypoxanthine guanine phosphoribosyltransferase or adenine phosphoribosyltransferase, the appropriate cell type may be tk-, hgprt- or aprt- cells, respectively. Or, normal cells can be used when the selectable marker is dhfr, gpt, neo or hygro, which confer resistance to methotrexate, mycophenolic acid, G-418 or hygromycin, respectively.

Antibody production

Antibodies that selectively recognize one or more epitopes of the peptides of this invention are also within the scope of the invention. Such antibodies include, for example, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, human antibodies, single chain antibodies, Fab fragments, F (ab ') ₂ fragments, molecules obtained using a library expressing Fab, human antibodies (polyclonal or monoclonal ) produced in transgenic mice and epitope-binding fragments of any of the above antibodies.

Antibodies can be used in conjunction with gene therapy methods to evaluate, for example, the expression of the peptides of this invention either in the cells or directly in the patient’s tissues into which the genes have been introduced.

To obtain antibodies, many host animals can be immunized by injection of the peptides of this invention, an antipeptide antibody, an antibody against a peptide analogue, or immunogenic fragments thereof by methods well known in the art. In the case of an anti-idiotypic antibody, the immunogen is an antibody to a peptide or an antibody to an analog of a peptide. The preparation of anti-idiotypic antibodies is described, for example, in US Pat. 4,699,880. Suitable host animals include, but are not limited to, rabbits, mice, goats, sheep, and horses. Methods of immunization are well known in the art. Polyclonal antibodies can be purified from the serum of immunized animals, or monoclonal antibodies can be prepared by methods well known in the art. These methods include, but are not limited to, the well-known hybridoma methods of Kohler and Milstein, human hybridoma B-cell based methods, and the EBV hybridoma method. Monoclonal antibodies can be any class of immunoglobulins, including IgG, IgE, IgM, IgA and IgD, containing light chains of either kappa or lambda. Methods for the preparation and use of chimeric antibodies are known in the art and are described, for example, in US Pat. 5807715, 4816397, 4816567, 5530101, 5585089, 5693761, 5693762, 6180370 and 5824307.

Tests for determining selectivity for CRF ₂ R

The pharmacological activity and selectivity of the peptides according to this invention can be determined using published testing methods (see, for example, application for the grant of US patent No.09 / 799978). Since CRF ₂ R and CRF ₁ R are homologous proteins, it is assumed that some of the CRF ₂ R agonists will also function as CRF ₁ R agonists. As discussed above, activation of CRF ₁ R induces activation of the HPA axis, resulting in increased production of corticosteroids leads to atrophy of skeletal muscles. In most cases where an increase in muscle mass or function is required, it is undesirable to activate the HPA axis. When selecting a peptide useful for treating a modulated CRF ₂ R disorder that is not associated with muscular dystrophy, it is preferred that the peptide is selective for CRF ₂ R. Preferably, the peptide exhibits 10-fold selectivity for CRF ₂ R compared to CRF ₁ R (i.e., 10 times more active with respect to CRF ₂ R than with respect to CRF ₁ R), more preferably 100-fold selectivity and most preferably 1000-fold or greater selectivity. Since published studies have shown the benefits of corticosteroid therapy in treating muscular dystrophies, it may be useful for the CRF ₂ R agonist to maintain some level of agonism with respect to CRF ₁ R when used for the treatment of muscular dystrophies. Thus, for the treatment of muscular dystrophies, a low selectivity peptide that activates CRF ₂ R as well as CRF ₁ R within similar concentration ranges is preferred. Preferably, the peptide has 100-fold selectivity for CRF ₂ R compared to CRF ₁ R, more preferably 10-fold selectivity and most preferably does not have selectivity for CRF ₂ R compared to CRF ₁ R (i.e., the activity of the selected for research, the compounds are essentially the same with respect to CRF ₂ R and CRF ₁ R). Also in this case, it may be preferable that the peptide is a full CRF ₂ R agonist and at the same time a partial CRF ₁ R agonist. Such a peptide, therefore, may have its inherent limitation on the maximum degree of increase in cortisol levels and the possibility of muscle atrophy, whereas the action against atrophy modulated through CRF ₂ R can be enhanced with increasing dose. _One skilled in the art can readily determine whether a peptide is a full or partial CRF ₁ R agonist or CRF ₂ R agonist using methods known in the art.

Since it is desirable to distinguish between binding to CRF ₂ R compared to CRF ₁ R, the assays described above can be performed using a cell or cell membrane that expresses only CRF ₂ R, or assays can be performed with a recombinant source of CRF ₂ R. Cells expressing both forms CRFR, may be modified using homologous recombination to inactivate or otherwise disable the CRF ₁ gene of R. Alternatively, if the source of CRFR contains more than one CRFR type, the background signal generated by the receptor which is not of interest, q.s. imo subtracted from the signal obtained in the assay. The background signal can be determined in a number of ways, including the exclusion of a signal from CRFR, which is not of interest, using an antisense sequence, antibodies, or selective antagonists. Known CRFR antagonists include, but are not limited to, antalarmin (selective for CRF ₁ R), antisauvagin-30 (selective for CRF ₂ R), and astressin (not selective for CRF ₁ R / CRF ₂ R).

To determine whether the peptide activates CRF ₂ R and / or CRF ₁ R, assays are usually based on cells; however, cell-free systems are known that are capable of differentiating the binding of an agonist and an antagonist, as described above. Cell-based assays include the steps of contacting cells that express CRF ₁ R or CRF ₂ R with a peptide of the invention or control and measure CRFR activation by measuring the expression or activity of components of signal transduction pathways involving CRFR.

As described in the Prerequisite section above, CRFRs appear to be conjugated via several different pathways, including Gαs, Gαq, or Gαi, depending on the type of cell. It is assumed that activation of CRFR by an agonist ensures signal transmission by the receptor through any of these pathways, provided that the necessary components of this pathway are present in a particular cell type. Thus, in order to analyze a particular peptide of the invention with respect to CRFR activation, any of the signal transduction pathways can be used as an indicator in the assay, even if CRFR is coupled with skeletal muscle atrophy in a suitable cell type for in vivo treatment by another pathway. One skilled in the art will recognize that the assay can be effective in identifying applicable peptide agonists, regardless of the way in which receptor activation is measured. Assays for measuring activation of these signaling pathways are known in the art.

For example, after making contact with the peptide of the invention, cell lysates can be obtained and analysis of cAMP induction can be performed. cAMP is induced in response to activation of Gαs. Since Gαs are activated by receptors other than CRFR, and since the test peptide can exert its effect through CRFR or through another mechanism, two control comparisons are appropriate to determine if the peptide increases cAMP levels by activating CRFR. In one control, cAMP levels of cells in contact with a peptide are compared with cAMP levels of cells in contact with a control compound (i.e., vehicle in which the peptide is dissolved). If the peptide increases cAMP levels compared to the control compound, this indicates that the peptide increases cAMP by some mechanism. In another control, cAMP levels are compared for a cell line expressing CRFR and a cell line that is essentially the same, except that it does not express CRFR, with both cell lines being treated with a peptide. If a peptide increases cAMP levels in a cell line expressing CRFR compared to a cell line that does not express CRFR, this is an indication that the peptide increases cAMP levels by activating CRFR.

In one example, cAMP induction is measured using DNA constructs containing a cAMP response element associated with any of a variety of reporter genes that can be introduced into cells expressing CRFR. Such reporter genes include, but are not limited to, chloramphenicol acetyl transferase (CAT), luciferase, glucuronide synthetase, growth hormone, fluorescent protein (e.g., green fluorescence protein) or alkaline phosphatase genes. After exposure of the cells with the peptide, the expression level of the reporter gene can be quantified to determine the ability of the peptide to increase cAMP levels and thus determine the ability of the peptide to activate CRFR.

The cells used in this assay are the same as the cells for the CRFR binding assay described above, except that the cells used in the activation assay preferably express a functional receptor that provides a statistically significant response to CRF or one or more analogs CRF In addition to using cells expressing full-length CRFRs, cells can be constructed that express CRFRs containing a ligand-binding receptor domain that is coupled to reporter elements or physically modified to contain reporter elements or to interact with signal-transmitting proteins. For example, wild-type CRFR or a CRFR fragment can be fused to a G protein, which leads to activation of the fused G protein when the agonist binds to the CRFR part of the fusion protein. Siefert, R. et al., Trends Pharmacol. Sci., 20: 383-389 (1999). Cells should also preferably have a number of parameters depending on the indicator system in order to maximize the response induced by CRF or an analogue of CRF, for example, to detect strong induction of the CRE reporter gene; (a) low natural levels of cAMP may be preferred; (b) G proteins capable of interacting with CRFR; (c) high levels of adenylyl cyclase; (d) high levels of protein kinase A; (e) low phosphodiesterase levels; and (f) a high level of a protein that binds a cAMP response element. To enhance the response to CRF or an analogue of CRF, host cells can be designed to express more preferred factors or fewer non-preferred factors. In addition, alternative CRE reporter induction pathways can be eliminated to reduce basal levels.

Assays for determining pharmacological activity

The pharmacological activity of the peptides according to this invention can be determined using published testing methods. For example, skeletal muscle atrophy or hypertrophy models include both in vitro cell culture models and in vivo skeletal muscle atrophy models in animals.

In vitro skeletal muscle atrophy models are known in the art. Such models are described, for example, in Vandenburgh, H. H., In Vitro, 24: 609-619 (1988), Vandenburgh, H. H., et al., J. Biomechanics, 24 Suppl. 1: 91-99 (1991), Vandenburgh, H. H., et al., In Vitro Cell. Dev. Biol., 24 (3): 166-174 (1988), Chromiak, J. A., et al., In Vitro Cell. Dev. Biol. Anim., 34 (9): 694-703 (1998), Shansky, J., et al., In Vitro Cell. Dev. Biol. Anim., 33 (9): 659-661 (1997), Perrone, C. E., et al., J. Biol. Chem., 270 (5): 2099-2106 (1995), Chromiac, J. A. and Vandenburgh, H. H., J. Cell. Physiol., 159 (3): 407-414 (1994), and Vandenburgh, H. H. and Karlisch, P., In Vitro Cell. Dev. Biol., 25 (7): 607-616 (1989).

A number of animal skeletal muscle atrophy models are known in the art, such as those described in the following publications: Herbison, G. J., et al. Arch. Phys. Med. Rehabil., 60: 401-404 (1979), Appell, H-J. Sports Medicine 10: 42-58 (1990), Hasselgren, P-O. and Fischer, J. E. World J. Surg., 22: 203-208 (1998), Agbenyega, E. T. and Wareham, A. C. Comp. Biochem. Physiol., 102A: 141-145 (1992), Thomason, D. B. and Booth, F. W. J. Appl. Physiol. 68: 1-12 (1990), Fitts, R. H., et al. J. Appl. Physiol., 60: 1946-1953 (1986), Bramanti, P., et al. Int. J. Anat. Embryol. 103: 45-64 (1998), Cartee, G. D. J. Gerontol. A Biol. Sci. Med. Sci., 50: 137-141 (1995), Cork, L. C., et al. Prog. Clin. Biol. Res., 229: 241-269 (1987), Booth, F. W. and Gollnick, P. D. Med. Sci. Sports Exerc., 15: 415-420 (1983), Bloomfield, S. A. Med. Sci. Sports Exerc., 29: 197-206 (1997). Preferred animals for these models are mice and rats. Said models include, for example, cessation of atrophy models, such as splinting or otherwise immobilizing limbs, suspending the hind limb, completely immobilizing the animal, and lower gravity. Models of atrophy induced by nerve damage include, for example, nerve crushing, removal of nerve regions that innervate specific muscles, application of a toxin to nerves, and infection of nerves with viral, bacterial, or eukaryotic infectious agents. Models of glucocorticoid-induced atrophy include the use of atrophic-inducing doses of exogenous glucocorticoid in animals and the stimulation of endogenous corticosteroid production, for example, using hormones that activate the hypothalamic-pituitary-adrenal (HPA) axis. Models of sepsis-induced atrophy include, for example, inoculation with sepsis-inducing organisms, such as bacteria, treating the animal with immune-activating compounds, such as bacterial cell wall extract or endotoxin, and piercing intestinal walls. Models of cachexia-induced atrophy include, for example, inoculating an animal with tumor causing cells with cachexia-forming potential, infecting the animal with infectious agents (such as viruses that cause AIDS) that result in cachexia, and treating the animal with hormones or cytokines, such as CNTF, TNF, IL-6, IL-1, etc., which induce cachexia. Models of atrophy induced by heart failure include treating the animal so that heart failure occurs with concomitant atrophy of skeletal muscle. Models of atrophy induced by a neurodegenerative disease include models of autoimmune diseases, such as models resulting from immunization of an animal with components of neurons. Muscle dystrophy-induced atrophy models include natural or artificial genetically induced muscular dystrophy models, such as a dystrophin gene mutation that occurs in Mdx mice.

Animal models of skeletal muscle hypertrophy include, for example, models of increased load on limb muscles resulting from inactivation of the opposite limb, reuse after an event that induces atrophy due to cessation of use, reuse of a muscle that is atrophied due to temporary nerve damage, increased use of individual muscles due to muscle inactivation synergist (for example, compensatory hypertrophy), increased muscle use as a result of increased load Ki placed on the muscle, and hypertrophy as a result of removal of the glucocorticoid after glucocorticoid-induced atrophy. Preferred animal atrophy models include the sciatic nerve denervation atrophy model, the glucorticoid-induced atrophy model, and the cessation of atrophy model by splinting the paw, which are described in more detail below.

The model of atrophy in the sciatic nerve denervation is to anesthetize the animal, followed by surgical removal of a short section of either the right or left sciatic nerve, for example, in mice, the sciatic nerve is isolated at approximately midpoint along the thigh and a section of approximately 3-5 mm is removed. This leads to denervation of the lower muscles of the hind limb, the result of which is atrophy of these muscles. Usually, the innervation of the biceps of the thigh is left intact to provide sufficient movement in the knee for almost normal walking. Typically, in untreated animals, denervated muscle mass is reduced by 30–50% ten days after denervation. After denervation, test peptides are administered, for example, by injection or continuous infusion, for example, by implantation of an osmotic mini-pump (e.g., Alzet, Palo Alto, CA) to determine their effect on denervation-induced atrophy of skeletal muscle. At various time points after denervation, animals kill and quickly cut out the lower part of the limb muscle on both denervated and non-denervated paws, muscles cleared of tendons and connective tissue are weighed. The degree of atrophy of the affected muscles is analyzed, for example, by measuring muscle mass, the cross-sectional area of the muscle, the cross-sectional area of myofibrils or the content of contractile protein.

A model of glucocorticoid-induced atrophy is the administration of a glucocorticoid to a test animal, for example, 1.2 mg / kg / day of dexamethasone in drinking water. Typically, in non-treated animals, skeletal muscle mass decreases by 30-50% ten days after dexamethasone administration. Along with or after administration of glucocorticoid, test peptides are administered, for example, by injection or continuous infusion, to determine their effect on glucocorticoid-induced atrophy of skeletal muscle. At various time points after glucocorticoid administration, the degree of atrophy in the affected muscles is analyzed, as described above for the denervation model.

The model of atrophy resulting from cessation of use when the tire is placed on the paw consists in laying the tire on one hind paw of the animal from the knee down through the foot. Usually muscle mass is reduced by 20-40% ten days after the application of the tire. Following application of the tire, test peptides are administered by injection or continuous infusion by implantation of an osmotic mini-pump (e.g., Alzet, Palo Alto, CA) to determine their effect on skeletal muscle atrophy induced by the application of the tire to the paw. At various time points after applying the tire to the paw, the degree of atrophy of the affected muscles is analyzed as described above for the denervation model.

The activity of test peptides in relation to bone can be easily demonstrated using an assay designed to test the ability of test compounds to increase bone volume, mass or density. An example of such an analysis is an analysis on ovariectomized rats.

When analyzing rats subjected to ovariectomy, the rats are removed from the ovaries of six months of age, kept for another 2 months and then a dose of the test compound is administered subcutaneously once a day. After completion of the study, bone mass and / or density can be measured by dual energy X-ray absorptiometry (DXA), or by peripheral quantitative computed tomography (pQCT), or computer microtomography (mCT). Alternatively, static and dynamic histomorphometry can be used to measure the increase in volume or bone formation.

Songs

Another aspect of the invention are compositions which comprise: (a) a safe and effective amount of a peptide according to the invention; and (b) a pharmaceutically acceptable carrier. Use standard methods for preparing pharmaceutical compositions, such as those described in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., Latest revision.

"Safe and effective amount" means the amount of the peptide according to the invention, sufficient to significantly induce a positive modification of the condition being treated, but small enough to avoid serious side effects (such as toxicity, irritation or allergic reaction) in the animal, preferably a mammal, more preferably a human in need thereof, corresponding to a reasonable benefit / risk ratio when used according to this invention july Obviously, the specific “safe and effective amount” will vary depending on factors such as the particular condition being treated, the physical condition of the subject, the duration of treatment, the nature of the concomitant therapy (if any), the particular dosage unit used, the vehicle used, the solubility of the peptide therein and the dosage schedule desired for the composition. One of skill in the art can use the following guidelines to determine the “safe and effective amount” of this invention (Spilker B., Guide to Clinical Studies and Developing Protocols, Raven Press Books, Ltd., New York, 1984, pp. 7 -13, 54-60; Spilker B., Guide to Clinical Trials, Raven Press, Ltd., New York, 1991, pp. 93-101; Craig C., and R. Stitzel, eds., Modern Pharmacology, 2d ed ., Little, Brown and Co., Boston, 1986, pp. 127-33; T. Speight, ed., Avery's Drug Treatment: Principles and Practice of Clinical Pharmacology and Therapeutics, 3d ed., Williams and Wilkins, Baltimore, 1987 , pp. 50-56; R. Tallarida, R. Raffa and P. McGonigle, Principles in General Pharmacology, Springer-Verlag, New York, 1988, pp. 18-20).

In addition to this peptide, the compositions of this invention contain a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable carrier” as used herein means one or more compatible solid or liquid excipients, diluents or encapsulating substances that are suitable for administration to an animal, preferably a mammal, more preferably a human. The term "compatible" as used in this description means that the components of the composition can be mixed with this peptide and with each other so that there is no interaction that can significantly reduce the pharmaceutical efficacy of the composition in normal use. Pharmaceutically acceptable carriers should of course be of a sufficiently high degree of purity and sufficiently low toxicity so that they are suitable for administration to an animal, preferably a mammal, more preferably a person being treated.

Some examples of substances that can serve as pharmaceutically acceptable carriers or their components are: sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and methyl cellulose; powdered tragacanth gum; malt; gelatin; talc; solid lubricants, such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cocoa butter; polyols such as propylene glycol, glycerin, sorbitol, mannitol and polyethylene glycol; alginic acid; emulsifiers such as Twins®; humectants such as sodium lauryl sulfate; dyes; flavoring agents; tabletting agents, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic salt solution and phosphate buffer solutions.

The choice of a pharmaceutically acceptable carrier used with this compound is mainly determined by the route of administration of the peptide.

If this peptide needs to be injected, the preferred pharmaceutically acceptable carrier is a sterile saline solution with a colloidal suspending agent compatible with blood, the pH of which has been adjusted to about 7.4.

In particular, pharmaceutically acceptable carriers for systemic administration include sugars, starches, cellulose and its derivatives, malt, gelatin, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffer solutions, emulsifiers, isotonic salt solution and pyrogen-free water. Preferred carriers for parenteral administration include propylene glycol, ethyl oleate, pyrrolidone, ethanol and sesame oil. Preferably, the pharmaceutically acceptable carrier in the compositions for parenteral administration is at least about 90% of the mass. total composition.

The compositions of this invention are preferably supplied in unit dosage form. As used herein, a “unit dosage form” is a composition of the invention comprising an amount of a peptide of formula (I) that is suitable for administration to an animal, preferably a mammal, more preferably a human, in a single dose in accordance with good medical practice. These compositions preferably contain from about 0.1 mg (milligram) to 1000 mg, more preferably from about 0.5 to 500 mg, more preferably from about 1 to 30 mg, peptides of formula (I).

The compositions of this invention may take any of a variety of forms suitable, for example, for oral, rectal, topical, nasal, ocular or parenteral administration. Depending on the particular route of administration desired, a variety of pharmaceutically acceptable carriers well known in the art can be used. These carriers include solid or liquid fillers, diluents, hydrotropic substances, surfactants and encapsulating substances. Optionally, pharmaceutically active products may be included that do not substantially interfere with the agonistic activity of the peptides of formula (I) with respect to CRF ₂ R. The amount of carrier used with the peptide of formula (I) is sufficient to provide a rational amount of product for administration to unit dose of a peptide of formula (I). Methods and compositions for preparing dosage forms applicable to the methods of this invention are described in the following publications: Modern Pharmaceutics, Chapters 9 and 10 (Banker & Rhodes, editors, 1979); Lieberman et al., Pharmaceutical Dosage Forms: Tablets (1981); and Ansel, Introduction to Pharmaceutical Dosage Forms 2d Edition (1976).

Various oral dosage forms may be used, including solid forms such as tablets, capsules, granules, and bulk powders. Said oral forms contain a safe and effective amount of the peptide, usually at least about 5% and preferably from about 25 to 50%. The tablets may be compressed, powdered, enteric coated, sugar coated, film coated, or complex compressed tablets containing suitable binders, lubricants, diluents, disintegrating agents, colorants, flavoring agents, passage inducing agents, and melting agents . Liquid oral dosage forms include aqueous solutions, emulsions, suspensions, solutions and / or suspensions, reconstituted from non-effervescent granules, and effervescent preparations, reconstituted from effervescent granules, and containing suitable solvents, preservatives, emulsifiers, suspending agents, diluents, sweeteners, melting agents , dyes and flavoring agents.

Pharmaceutically acceptable carriers suitable for the preparation of oral dosage forms are well known in the art. Tablets typically contain, as inert diluents, conventional pharmaceutically compatible adjuvants such as calcium carbonate, sodium carbonate, mannitol, lactose and cellulose; binders such as starch, gelatin and sucrose; disintegrating agents such as starch, alginic acid and croscarmellose; lubricants such as magnesium stearate, stearic acid and talc. Glidants, such as silica, can be used to improve the flow characteristics of the powder mixture. Dyes such as FD&C dyes can be added for appearance. Sweeteners and flavoring agents such as aspartame, saccharin, menthol, mint and fruit flavoring are useful as adjuvants for chewable tablets. Capsules typically contain one or more of the solid diluents described above. The selection of carrier components depends on secondary criteria, such as taste, cost and shelf life, which are not critical for the purposes of this invention and can easily be carried out by a person skilled in the art. In general, the preparation will contain a peptide and inert ingredients that provide protection against the environment in the stomach and the release of the biologically active product in the intestine.

The peptide of formula (I) can be chemically modified so that the oral delivery of the derivative is effective. In general, the intended chemical modification is the binding of at least one moiety to the protein molecule itself, the moiety providing: (a) inhibition of proteolysis; and (b) absorption into the blood stream from the stomach or intestines. It is also desirable to increase the overall stability of the protein and increase circulation time in the body. Examples of such constituents include: polyethylene glycol, ethylene glycol-propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone and polypropylene (Newmark et al., J. Appl. Biochem., 4: 185-189 (1982)). Other polymers that can be used are poly-1,3-dioxolane and poly-1,3,6-thioxoxane. Preferred for pharmaceutical use as described above are polyethylene glycol constituents.

The release site may be the stomach, small intestine (duodenum, jejunum or ileum) or large intestine. The person skilled in the art has compositions that will not dissolve in the stomach, but will release material in the duodenum or elsewhere in the intestine. Preferably, the release will not be affected by the environment in the stomach by protecting the peptide (or variant) or by releasing the biologically active material outside the stomach, namely in the intestine.

To ensure complete stability in the stomach, preferably a coating impermeable at least at pH 5.0. Examples of the most common inert ingredients that are used as enteric coatings are cellulose acetate trimellitate (CAT), hydroxypropyl methylcellulose phthalate (HPMCP), HPMCP 50, HPMCP 55, polyvinyl acetate phthalate (PVAP), euradit L30D, Aquateric cellulose, phthalate , erases L, erases S and shellac. These coatings can be used as mixed films.

Oral compositions also include liquid solutions, emulsions, suspensions, and the like. Pharmaceutically acceptable carriers suitable for preparing such compositions are well known in the art. Common carrier components for syrups, elixirs, emulsions and suspensions include ethanol, glycerol, propylene glycol, polyethylene glycol, liquid sucrose, sorbitol and water. In the case of suspensions, conventional suspending agents include methyl cellulose, sodium carboxymethyl cellulose, Avicel® RC-591, tragacanth gum and sodium alginate; typical humectants include lecithin and polysorbate 80; and common preservatives include methyl paraben and sodium benzoate. Oral liquid compositions may also contain one or more components, such as sweeteners, flavoring agents and coloring agents, as discussed above.

The compositions of this invention may optionally contain other active agents. Non-limiting examples of active agents are listed in WO 99/15210.

Other compositions useful for achieving systemic delivery of these compounds include sublingual, buccal, suppository, nasal and pulmonary dosage forms. Such compositions typically contain one or more soluble excipients, such as sucrose, sorbitol and mannitol; and binders such as gum arabic, microcrystalline cellulose, carboxymethyl cellulose and hydroxypropyl methyl cellulose. Glidants, lubricants, sweeteners, colorants, antioxidants and flavoring agents described above may also be included.

The compositions of this invention can also be administered topically to a subject, for example, by direct application or spreading of the composition onto the epidermal or epithelial tissue of the subject or transdermally using a “patch”. An example of a suitable patch applicator is described in U.S. Patent Application No. 10/054113. Such compositions include, for example, lotions, creams, solutions, gels and solids. These topical compositions preferably contain a safe and effective amount, usually at least about 0.1% and preferably about 1 to 5% of the peptide. Suitable carriers for topical administration are preferably stored on the skin as a continuous film and are resistant to sweat removal or immersion in water. Typically, the carrier is organic in nature and the peptide can be dispersed or dissolved in it. The carrier may contain pharmaceutically acceptable emollients, emulsifiers, thickeners, solvents, and the like.

Administration Methods

The invention also relates to methods for treating modulated CRF ₂ R disorders in a human or other animal subject by administering a safe and effective amount of the peptide to said subject. The methods of the invention are useful for the prophylaxis or treatment of the disorders described above.

The compositions of this invention may be administered topically or systemically. Systemic use includes methods of introducing a peptide of formula (I) into body tissues, for example, into the joints (especially in the treatment of rheumatoid arthritis), intrathecal, epidural, intramuscular, transdermal, intravenous, intraperitoneal, subcutaneous, nasal, pulmonary, sublingual, rectal and oral administration .

The specific dose of the peptide to be administered, as well as the duration of the treatment and whether the treatment is local or systemic, depend on each other. The dose and treatment regimen will also depend on factors such as the particular peptide used, indication for treatment, the ability of the peptide to achieve minimal inhibitory concentrations at the site of tissue to be processed, the subject’s personal characteristics (such as weight), compliance with the treatment regimen and availability, and the severity of any side effects of the treatment.

Topical administration can be used to deliver the peptide systemically or to treat a subject locally. The amount of peptide for topical administration depends on factors such as the sensitivity of the skin, the type and position of the tissue being treated, the composition and carrier (if any) to be administered, the particular peptide administered, and the particular disorder being treated and the degree of systemic requirements ( which are different from local) effects.

The peptides of this invention can be targeted to specific target sites where treatment is needed using ligands directing to the target. For example, in order to direct the peptide to the treatment of muscular dystrophy, the peptide is conjugated to an antibody or fragment thereof that is immunoreactive with a skeletal muscle marker, which is commonly known in the art. The ligand directing the target may also be a ligand suitable for a receptor that is present on skeletal muscle. Any ligand directing to the target can be used that specifically interacts with the marker of the target tissue. Methods of binding a compound of the invention to a targeting ligand are well known and similar to the methods described below for binding to a carrier.

The peptide of formula (I) can be administered by controlled release. For example, a peptide can be administered using intravenous infusion, an implantable osmotic pump, a transdermal patch, liposomes, a subcutaneous depot injection containing biodegradable material, or other routes of administration. In one embodiment, a pump can be used (Langer et al., Eds., Medical Applications of Controlled Release, CRC Pres., Boca Raton, Fla. (1974); Sefton, CRC Crit. Ref. Biomed. Eng., 14: 201 ( 1987); Buchwald et al., Surgery 88: 507 (1980); Saudek et al., N. Engl. J. Med. 321: 574 (1989)). Alternatively, polymeric materials can be used (Langer, 1974, supra; Sefton, 1987, supra; Smolen et al., Eds., Controlled Drug Bioavailability, Drug Product Design and Performance, Wiley, NY (1984); Ranger et al., J. Macromol. Sci. Rev. Macromol. Chem., 23: 61 (1983); see also Levy et al., Science, 228: 190 (1985); During et al., Ann. Neurol., 25: 351 ( 1989); Howard et al., J. Neurosurg., 71: 105 (1989)). In yet another embodiment, a controlled release system can be placed near a therapeutic target, with only a portion of the systemic dose being required (see, for example, Goodson, Medical Applications of Controlled Release, vol. 2, pp. 115-138 (1984)). In yet another embodiment, a polymer-based drug delivery system is used in which drugs are delivered from a polymer or lipid systems. These systems deliver drugs by three common mechanisms: (1) diffusion of the type of drug from or through the system; (2) a chemical or enzymatic reaction leading to degradation of the system, or cleavage of the drug from the system; and (3) solvent activation, either through osmosis or swelling of the system. Suitable systems are described in review articles: Langer, Robert, Drug delivery and targeting, Nature: 392 (Supp): 5-10 (1996); Kumar, Majeti NV, Nano and Microparticles as Controlled Drug Delivery Devices, J. Pharm. Pharmaceut. Sci., 3 (2): 234-258 (2000); Brannon-Peppas, "Polymers in Controlled Drug Delivery", Medical Plastics and Biomaterials, (Nov. 1997). See also Langer, 1990, supra; Treat et al., In Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Langer, Science, 249: 1527-1533 (1990). Suitable systems may include: an Atrigel ^TM drug delivery system from Atrix Labs; DepoFoam ^TM from SkyPharma; polyethylene glycol based hydrogels from Infimed Therapeutics, Inc .; solubilizing delivery system ReGel ^TM medicament, SQZGel ^TM oral, HySolv ^TM and ReSolv ^TM from MacroMed; ProGelz ^TM from ProGelz 'Products; and injection ProLease ^TM from Alkermes.

Of course, in all of the above cases, the peptides according to the invention can be entered separately or in the form of mixtures, and the composition, in addition, may contain additional drugs or excipients that correspond to the indication.

Gene therapy

Expression vectors can be used to introduce the nucleic acids of the invention into a cell as part of gene therapy. Such vectors typically have suitable restriction sites located close to the promoter sequence to allow insertion of nucleic acid sequences. Transcriptional cassettes can be prepared containing a transcription initiation region, a target gene or fragment thereof, and a transcription termination region. Transcriptional cassettes can be inserted into many vectors, for example, a plasmid, retrovirus, lentivirus, adenovirus, and the like, since the vectors are able to temporarily or stably remain in cells, usually for a period of at least one day, more usually for a period of time of at least a few days to several weeks.

The proteins and nucleic acids of the invention can be introduced into tissues or host cells by any of a number of methods, including viral infection, microinjection, or fusion of vesicles. Needle-free injection for intramuscular administration can also be used as described by Furth et al., Anal. Biochem. 205: 365-368 (1992). It is possible to coat DNA gold microparticles and deliver intradermally using a particle bombardment device or “gene gun” as described in the literature (see, for example, Tang et al., Nature 356: 152-154 (1992), where gold microparticles cover DNA , then bombarded into skin cells).

Sets

It is within the scope of this invention to provide a kit for the prevention or treatment of a modulated CRF ₂ R disorder, comprising: (a) a peptide of formula (I) in a dosage form; and (b) instructions for use. Such a kit preferably contains a number of dosage units. Such kits may contain a fee with doses arranged in the order of their intended use. For example, such a kit may be a “blister pack”. Blister packs are well known in the packaging industry and are widely used for packaging pharmaceutical dosage forms. If desired, a means for memory may be provided, for example in the form of numbers, letters or other marks or with a calendar insert, where days are indicated in the treatment regimen at which doses can be administered. An example kit is described in WO 01/45636. Treatment regimens are the responsibility of specialists in the field of medicine. Non-limiting examples include taking once daily, weekly, bi-weekly, monthly or bi-monthly.

EXAMPLES

Example 1

Sauvagin and other non-selective CRFR agonists are usually ineffective in the treatment of modulated CRF ₂ R disorders, as these agonists also activate CRF ₁ R, thereby leading to undesirable side effects.

Table 2 shows the comparative CRF binding for fragments of the native sequence of human urocortin I (hUcnI), human urocortin II (hUroII), human urocortin III (hUroIII), human corticotropin releasing factor (hCRF), sheep corticotropin (oCRF) and sauvagin (Svg ) designated as SEQ ID NO: 2, 4, 6, 8, 10, and 11, respectively.

table 2 SEQ ID NO Peptide E CRF ₂ R EC ₅₀ (nM) (Emax%) CRF ₁ R EC ₅₀ (nM) (Emax%) 2 hUcnI 3.52 (100) 9.00 (100) four hUroII 3.64 (98) > 100 (9) 6 hUroIII > 100 (60) > 1000 (10.25) 8 hCRF 49.25 (100) 19.95 (87) 10 oCRF > 100 (33) 27.35 (98) eleven Svg 6.03 (95) 17.60 (100)

Example 2

Table 3 shows the comparative binding of CRF for various variants of the invention.

Table 3 SEQ ID NO CRF ₂ R EC ₅₀ (nM) (Emax%) CRF ₁ R EC ₅₀ (nM) (Emax%) one 31.50 (100) 783 (64) 3 12.13 (88) 1000 (12) 5 100 (12) 100 (9) 7 100 (19) 100 (4) 9 100 (34) 100 (10) 12 9.72 (91) 928 (73) 13 3.88 (79) 97.65 (69) fourteen 6.33 (90) 109.00 (92) fifteen 6.56 (97) 85.30 (86) 16 7.88 (86) 136.00 (98) 17 10.20 (96) 260.50 (98) eighteen 5.29 (97) 106.00 (100) 19 7.42 (75) 232.50 (83) twenty 7.81 (99) 906.00 (60) 21 8.46 (96) 908.00 (88) 22 8.33 (100) 1000.00 (64) 23 10.20 (100) 1000.00 (82) 24 43.15 (100) > 1000 (18) 25 80.95 (81.90) 677.00 (92.35) 26 91.75 (86) > 1000 (16) 27 10.70 (96) 325 (100) 28 100 (15) > 1000 (2) 29th 16.00 (100) > 1000 (79) thirty 18.70 (85.50) 100.45 (100) 31 30.75 (100) > 1000 (18) 32 20.25 (98) 606 (94) 34 > 100 (45) > 1000 (11) 35 15.55 (88) > 1000 (74) 36 14.75 (73) > 1000 (33) 37 > 100 (51) > 1000 (5) 38 71.90 (91) > 1000 (11) 39 58.17 (94) 1000 (63) 40 6.95 (93) 102.5 (99) 41 18.30 (100) > 1000 (43) 42 > 100 (88) > 1000 (10) 43 > 100 (67) > 1000 (10) 44 19.15 (87) 943.50 (64) 45 > 100 (44) > 1000 (7) 46 > 100 (100) > 1000 (17) 47 > 100 (66) 1000 (12) 48 100 (14) 1000 (21) 49 > 100 (37) > 1000 (15) fifty 19.04 (94) > 1000 (42) 51 20.65 (100) > 1000 (48) 52 > 100 (10) > 1000 (13) 53 > 100 (95) > 1000 (19) 54 100 (11) 1000 (10) 55 7.95 (95) 11.60 (92) 56 50.35 (87) > 100 (14) 57 > 100 (46) > 100 (12) 58 71.60 (100) > 100 (16) 59 > 100 (27) > 100 (10) 60 > 100 (44) > 100 (8) 61 > 100 (89) > 100 (12) 63 67.35 (100) 73.15 (34) 64 63.30 (94) 68.90 (57) 65 67.90 (64) > 100 (16) 67 10.02 (50) 44.17 (96) 68 38.55 (74) > 100 (33) 69 5.85 (81) 34.50 (88) 70 18.25 (82) > 100 (8) 71 94.80 (56) > 100 (6) 72 > 100 (55) > 100 (4) 73 > 100 (11) > 100 (11) 74 54.97 (100) > 100 (7) 75 > 100 (52) > 100 (5) 76 91.45 (76) > 100 (7) 77 43.35 (100) > 100 (5) 78 24.65 (78) > 100 (6) 79 22.30 (100) > 100 (8) 80 6.53 (88) > 100 (55) 81 4.30 (73) 60.90 (81) 82 10.87 (90) 96.20 (85) 83 1.91 (81) 52.17 (96) 84 1.77 (100) 82.23 (99) 85 2.34 (100) 11.00 (84) 86 100 (8.10) 100 (4.60) 87 100 (14.65) 100 (5.30) 88 100 (12.60) 100 (11.15) 89 100 (12.70) 90 100 (12.25) 100 (4.00) 91 100 (7.10) 100 (4.00) 92 100 (15.85) 100 (4.60) 93 100 (6.40) 100 (5.00) 94 100 (6.15) 100 (7.30) 95 100 (8.25) 100 (5.55) 96 100 (12.50) 100 (16.30) 97 100 (7.60) 100 (4.25) 98 100 (5.50) 100 (4.00) 99 100 (4.35) 100 (4.35) one hundred 100 (9.85) 100 (6.25) 101 100 (6.95) 100 (7.35) 102 100 (13.50) 100 (7.80) 103 100 (4.85) 100 (5.75) 104 100 (4.50) 100 (11.10) 105 100 (9.15) 100 (5.20) 106 100 (6.10) 100 (4.80) 107 12.13 (87.90) 1000 (12.40) 108 79.00 (97) > 100 (3) 109 11.83 (91.67) 100 (6.70) 110 10.96 (100) > 100 (9) 111 10.95 (99) > 100 (9) 112 12.30 (100) > 100 (10) 113 11.30 (98) > 100 (4) 114 3.42 (100) > 100 (6) 115 13.60 (98) > 100 (7) 116 100 (26.45) 100 (4.80) 117 9.41 (98.85) 100 (7.80) 118 14.60 (100) > 100 (5) 119 3.57 (95) > 100 (3) 120 69.90 (100) 100 (7) 121 5.67 (91) > 100 (3) 122 3.31 (97) 1000 (10.7) 123 3.49 (93.75) > 1000 (9.60) 124 3.49 (94) > 100 (6) 125 4.47 (99) > 100 (9) 126 13.00 (91) > 100 (7) 127 7.79 (94) > 100 (6) 128 2.85 (98) > 100 (8) 129 3.83 (92) > 100 (12) 130 8.57 (92) > 100 (9) 131 5.25 (91) > 100 (8) 132 7.53 (88) > 100 (8) 133 12.22 (88) > 100 (4) 134 > 100 (19) > 100 (7) 135 > 100 (76) > 100 (6) 136 23.40 (68) > 100 (8) 137 36.90 (100) > 100 (4) 138 59.00 (46) > 100 (5) 139 42.60 (60) > 100 (4) 141 > 100 (29) > 100 (7) 142 9.08 (77.00) 43.45 (85.35) 143 11.05 (85.50) 232.00 (100) 144 9.16 (85.53) 567 (100) 145 7.80 (69.00) 196.50 (91.30) 146 8.20 (84.50) 103 (100) 147 6.75 (94.00) 101.60 (96.00) 148 9.45 (51.50) 295.00 (100) 149 26.20 (95.50) 1000 (40.70) 150 34.65 (70.50) 1000 (5.70) 151 36.75 (96.00) 1000 (19.90) 152 > 100 (19) > 100 (4) 153 9.28 (97) > 100 (7) 154 10.30 (100) > 100 (7) 155 20.60 (94) 40.65 (16) 156 9.29 (79) > 100 (6) 157 42.00 (60.65) 100 (91.00) 158 6.37 (89) > 100 (15) 159 90.77 (62) > 100 (9) 160 9.15 (87) > 100 (10) 161 > 100 (88) > 100 (77) 162 > 100 (7) > 100 (11) 163 4.49 (96) > 100 (13) 164 2.24 (92) > 100 (26) 165 > 100 (75) > 100 (19) 166 3.7 (99) 437.5 (95) 167 13.0 (100) 1000,0 (15) 168 4.9 (75) 1000,0 (52) 169 17.8 (93) 978.0 (26) 170 75.0 (83) 1000,0 (9) 171 17.6 (100) 206.0 (99) 172 13.5 (100) 1000,0 (68) 173 100 (65.20) 1000 (6.05) 174 4.42 (97.2) 1000.0 (29.10) 175 5.42 (92.15) 1000.0 (34.8) 176 12.60 (90.25) 1000.0 (26.9) 177 5.63 (97.65) 613.0 (69.2) 178 5.17 (96.45) 1000.0 (55.70) 179 10.22 (92.5) 477.0 (78.90) 180 3.14 (95.7) 125.0 (99.3) 181 5.22 (97.75) 154.0 (100) 182 7.21 (91.4) 409.0 (89.7) 183 7.93 (100) 415 (57.5) 184 3.72 (96.45) 486.0 (77.35) 185 8.98 (100) 358.5 (95.8) 186 25.05 (100) 323 (100) 187 10.3 (100) 31.6 (72) 188 14.6 (100) 162.5 (96) 189 7.0 (96) 62.2 (57) 190 39.60 (31) > 100 (9) 191 8.3 (100) 63.7 (65) 192 66.8 (97) 562.0 (99) 193 10.1 (97) 265.5 (95) 194 5.0 (96) 106.0 (94) 195 18.8 103.0 (93) 196 27.7 (97) 447.0 (96) 197 48.1 (94) 198 29.9 (100) 199 8.5 (92) 706.5 (93) 200 8.8 (100) 188.5 (99) 201 5.0 (100) 99.6 (80) 202 8.7 (99) 403.5 (100) 203 5.2 (94) 76.2 (86) 204 3.6 (93) 32.1 (74) 205 25.0 (93) 126.5 (88) 206 30.5 (97) 696.5 (97) 207 61.4 (96) 465.5 (88) 208 5.6 (88) 64.9 (81) 209 7.4 (93) 26.4 (80) 210 10.2 (97) 43.5 (90) 211 59.5 (100) 826.0 (37) 212 21.3 (100) 445.0 (100) 213 22.3 (99) 1000,0 (76) 214 74.30 (60) 100 (4) 215 4.0 (100) 187.5 (100) 216 9.9 (90) 49.8 (98) 217 4.7 (95) 94.7 (100) 218 4.8 (96) 98.4 (100) 219 7.8 (98) 80.9 (100) 220 4.1 (98) 63.6 (81) 221 8.5 (100) 236.5 (100) 222 9.4 (100) 384.5 (95) 223 3.0 (92) 48.1 (81) 224 26.9 (100) 1000,0 (27) 225 4.8 (89) 219.5 (97) 226 7.6 (100) 315.0 (95) 227 33.6 (95) 918.0 (18) 228 7.1 (100) 275.5 (100) 229 10.3 (100) 298.0 (100) 230 8.1 (100) 219.0 (100) 231 5.9 (100) 94.0 (100) 232 45.3 (100) 1000,0 (7) 233 46.1 (95) 1000 (11) 234 20.6 (100) 434.3 (96) 235 25.7 (100) 806.5 (70) 236 40.4 (97) 1000,0 (15) 237 22.2 (93) 1000,0 (18) 238 16.7 (100) 753.0 (88) 239 13.2 (100) 587.0 (80) 240 22.0 (100) 915.0 (80) 241 12.6 (99) 307.5 (99) 242 29.0 (94) 358.5 (99) 243 16.8 (100) 440.5 (96) 244 8.8 (98) 299.5 (100) 245 7.5 (93) 381.0 (100) 246 38.8 (100) 1000,0 (33) 247 18.8 (100) 1000,0 (34) 248 19.6 (98) 1000,0 (32) 249 12.2 (92) 1000,0 (50) 250 19.7 (100) 137.5 (99) 251 11.8 (100) 926.0 (80) 252 22.3 (100) 226.5 (100) 253 41.8 (86) 1000,0 (42) 254 100.0 (36) 708.0 (6) 255 7.0 (100) 33.3 (84) 256 12.6 (100) 253.5 (100) 257 100 (72.60) 744.50 (83.50) 258 100 (49.30) 1000 (23.65) 259 100 (64.95) 819.50 (83.60) 260 100 (89.35) 834.00 (89.70) 261 100 (95.30) 274 (100) 262 100 (92.25) 408 (100) 263 36.17 (93.03) 802.50 (71.95) 264 100.00 (66.30) 704.50 (100) 265 100.00 (23.35) 1000.00 (9.30) 266 100.00 (19.35) 1000.00 (4.45) 267 100.00 (44.20) 1000.00 (22.80) 268 100.00 (59.05) 1000.00 (14.15) 269 100.00 (77.30) 1000.00 (44.30) 270 100.00 (19.30) 1000.00 (7.85) 271 48.10 (68.95) 815.00 (80.65) 272 23.30 (100.00) 1000.00 (51.10) 273 31.30 (100.00) 1000.00 (59.30) 274 13.80 (100.00) 508.00 (80.90) 275 46.60 (100.00) 1000.00 (38.30) 276 22.10 (100.00) 1000.00 (75.70) 277 28.20 (100.00) 1000.00 (39.90) 278 19.55 (100.00) 1000.00 (48.70) 279 13.10 (100.00) 1000.00 (93.00) 280 100.00 (82.30) 1000.00 (11.80) 281 100.00 (78.80) 1000.00 (12.20) 282 25.80 (60.75) 1000.00 (21.75) 283 10.55 (71.95) 635.00 (100.00) 284 100.00 (100.00) 1000.00 (27.70) 285 13.95 (97.10) 1000.00 (11.10) 286 13.50 (92.45) 1000.00 (19.20) 287 11.31 (100.00) 1000.00 (51.75) 288 14.70 (100.00) 838.00 (31.65) 289 12.16 (97.30) 1000.00 (69.55) 290 100.00 (100.00) 1,000.00 (6.40) 291 100.00 (63.75) 1000.00 (4.75) 292 100.00 (86.10) 1000.00 (7.00) 293 40.30 (87.00) 520.50 (95.30) 294 100.00 (100.00) 1000.00 (13.60) 295 55.05 (67.60) 1000.00 (5.30) 296 6.66 (65) 100 (4) 297 100.00 (88.30) 1000.00 (27,250 298 82.00 (98.85) 1000.00 (20.05) 299 46.40 (71.55) 1000.00 (10.20) 300 17.10 (100.00) 1000.00 (7.95) 301 50.45 (88.40) 690.00 (84.10) 302 36.20 (100.00) 366.50 (100.00) 303 27.25 (100.00) 581.50 (100.00) 304 19.30 (92.55) 115.50 (100.00) 305 35.45 (95.20) 1000.00 (59.55) 306 27.55 (100.00) 608.00 (97.65) 307 5.82 (96.55) 78.40 (92.65) 308 3.30 (72.80) 63.45 (100.00) 309 5.55 (99.90) 107.50 (96.35) 310 8.70 (87.55) 1000.00 (45.30) 311 11.65 (100.00) 1000.00 (29.70) 312 14.05 (95.00) 869.50 (62.00) 313 11.05 (95.00) 704.00 (87,100 314 10.35 (99.75) 978.50 (82.40) 315 9.35 (81.70) 454.50 (100.00) 316 10.15 (94.50) 221.50 (92.35) 317 9.30 (88.35) 187.50 (100.00) 318 9.95 (95.40) 134.50 (92.85) 319 8.50 (95.00) 106.00 (88.30) 320 19.05 (100.00) 718.00 (68.75) 321 19.55 (86.80) 1000.00 (33.80) 322 23.05 (96.45) 1000.00 (10.65) 323 19.60 (100.00) 1000.00 (36.90) 324 17.20 (100.00) 1000.00 (46.60) 325 11.67 (100) 100 (5) 326 33.70 (100.00) 1000.00 (30.10) 327 28.40 (100.00) 1000.00 (36.50) 328 11.70 (95.70) 1000.00 (30.70) 329 5.15 (98.30) 1000.00 (98.40) 330 6.00 (93.65) 1000.00 (86.80) 331 9.85 (100.00) 1000.00 (78.65) 332 9.95 (100.00) 1000.00 (61.30) 333 9.85 (96.90) 1000.00 (43.80) 334 13.15 (93.55) 1000.00 (82.60) 335 28.05 (90.95) 1000.00 (49.45) 336 17.80 (100.00) 1000.00 (59.90) 337 23.95 (86.65) 1000.00 (36.45) 338 19.30 (77.55) 1000.00 (41.10) 339 100.00 (47.90) 1000.00 (13.20) 340 7.99 (100.00) 739.50 (95.95) 341 8.83 (95.50) 850.50 (82.35) 342 20.25 (92.25) 1000.00 (19.65) 343 13.60 (96.55) 783.00 (62.50) 344 4.30 (94.47) 650.00 (77.65) 345 39.70 (100.00) 1000.00 (18.75) 346 8.48 (97.75) 1000.00 (59.00) 347 22.35 (95.65) 1000.00 (48.75) 348 5.77 (90.40) 630.00 (86.05) 349 13.75 (100) 1000 (44.20) 350 11.59 (98.10) 1000 (48.00) 351 12.93 (97.37) 1000 (85.70) 352 8.26 (83.65) 780 (82.60) 353 4.75 (89.90) 229.50 (92.25) 354 6.48 (100) 1000 (13.40) 355 6.03 (95) 18 (100) 356 83.05 (16) 1000 (9) 357 6.44 (100) 331 (100) 358 5.56 (100) 99 (100) 359 23.10 (100) 230 (100) 360 6.12 (93) 157 (100) 361 6.37 (99) 149 (100) 362 4.39 (100) 386 (100) 363 25.15 (100) 1000 (20) 364 13.20 (100) 1000 (27) 365 23.45 (94) 1000 (21) 366 100.00 (46) 1000 (9) 367 100.00 (18) 1000 (6) 368 50.35 (91) 1000 (6) 369 100.00 (18) 1000 (4) 370 51.05 (75) 1000 (33) 371 6.62 (97) 1000 (22) 372 14.20 (100) 1000 (13) 373 11.54 (100 1000 (9) 374 15.75 (91) 1000 (11) 375 11.50 (100) 1000 (22) 376 52.55 (100) 1000 (8) 377 19.25 (83) 1000 (25) 378 14.88 (100) 1000 (36) 379 70.55 (94) 1000 (8) 380 19.00 (100) 1000 (16) 381 12.73 (99) 1000 (27) 382 39.45 (100) 1000 (8) 383 9.31 (96) 1000 (55) 384 7.10 (97.30) 1000 (70.30) 385 10.25 (100) 1000 (46) 386 8.70 (96) 1000 (78.25) 387 17.85 (100) 1000 (50.10)

Example 3

Increased In vivo Efficiency

The peptides according to this invention exhibit increased bioavailability, especially at low dose conditions, compared with known native sequences, for example a fragment of the UroII peptide (SEQ ID NO: 4).

The half-life of a peptide in a subject can be determined, for example, by high performance liquid chromatography (HPLC) of serum samples collected from the subject at various time points after administration of the peptide. One skilled in the art will know how to select suitable elution buffers for HPLC based on the physicochemical properties of a particular peptide.

A non-limiting example of an in vivo study to determine efficacy is provided herein. A dose of a peptide of formula (I) is administered to mice by the intravenous (iv) (1000 μg / kg) and subcutaneous (s / c) (1000 μg / kg) routes. At various time points after the dose (iv = 0, 2, 10, 30 min and 1, 2, 4 and 6 hours; and s / c = 0; 0.25; 0.5; 1; 2; 4 and 6 hours) receive blood samples in microcentrifuge tubes containing sodium heparin. Then, blood samples are processed to obtain plasma, which is stored at -70 ⁰ C until analysis.

Get plasma standards. On the day of analysis, an injection solution of the peptide of formula (I) is prepared in a concentration range from 50 ng / ml to 100 μg / ml in methanol by serial dilution of a pre-prepared stock methanol solution of the peptide of formula (I) at a concentration of 1 mg / ml. Similarly, on the day of analysis, an internal standard injection solution (ISTD) labeled with the stable h-Unc-II isotope is prepared by serial dilution of the stored 1 mg / ml ISTD stock solution to obtain a final concentration of 5 μg / ml. Working plasma standards are prepared in the weight range from 0.5 to 100 ng by adding 10 μl of the injection solution of the corresponding peptide of formula (I) to tubes already containing 10 μl of 5 μg / ml ISTD solution, 100 μl of dd-water and 100 μl of control plasma rats. Working standards are prepared for analysis as described below.

Prepare samples for quality control (QC). The initial QC solution is prepared at a concentration level of 50 ng / ml by adding 25 μl of an injection solution of the peptide of formula (I) with a concentration of 1 μg / ml in 475 μl of rat heparinized control plasma in a plastic bottle. Working QC samples are prepared by adding 100 μl of the original QC solution (50 ng / ml) to tubes already containing 10 μl of ISTD solution with a concentration of 5 μg / ml and 100 μl of dd-water. A working QC sample is prepared for analysis as described below.

Prepare test samples. On the day of analysis, the samples were thawed at room temperature and an aliquot of the sample was added to a tube already containing 10 μl of a 5 μg / ml ISTD solution, 100 μl of dd water and an aliquot of control rat heparinized plasma. The volume of the sample and the control plasma of rats is such that the total plasma volume is 100 μl.

Working standards, working QC samples and test samples are prepared for analysis by adding 400 μl of acetonitrile to tubes, each of which contains these samples, is closed, shaken, centrifuged and the supernatant is isolated. An aliquot (300 μl) of the supernatant was dried under N ₂ and redissolved in 50 μl of a methanol / water mixture (50/50).

Prepared working standards, working QC samples and test samples are analyzed by separation using gradient reverse phase high performance liquid chromatography (RP-HPLC) followed by electrospray ionization of the sample (ESI) with detection by mass spectroscopy / mass spectroscopy (MS / MS) using selective reaction monitoring (SRM) in positive ion mode. In the case of h-Unc-II and ISTD control the SRM channel.

Dose solutions from a pharmacokinetic study are diluted with methanol and analyzed by RP-HPLC with UV registration. The concentration of the peptide of formula (I) in dose solutions is calculated by interpolating a linear regression curve obtained on the basis of known standards.

Although specific embodiments of the invention have been illustrated and described, it will be understood by those skilled in the art that various changes and modifications can be made without departing from the gist and without departing from the scope of the invention. Therefore, it is intended that the appended claims cover all such changes and modifications that are within the scope of this invention.

Claims (19)

1. Non-native peptide according to the formula: IVLSLDVPIGLLQILLEQX ₁₉ KX ₂₁ X ₂₂ X ₂₃ X ₂₄ X ₂₅ X ₂₆ QATTNARILARV (SEQ ID N0: 531) (a) X _{19 is} selected from the group consisting of D and E; (b) X _{21 is} selected from the group consisting of A and Q; (c) X _{22 is} selected from the group consisting of R, E, and K; (d) X _{23 is} selected from the group consisting of A, K, and N; (e) X _{24 is} selected from the group consisting of A, E, and L; (f) X _{25 is} selected from the group consisting of R and K; and (g) X _{26 is} selected from the group consisting of E and Q. 2. The peptide according to claim 1, where X ₁₉ represents D. 3. The peptide according to claim 1, where X ₂₁ represents A. 4. The peptide according to claim 1, where X ₂₂ represents R. 5. The peptide according to claim 1, where X ₂₂ represents E. 6. The peptide according to claim 1, where X ₂₃ represents A. 7. The peptide according to claim 1, where X ₂₃ represents K. 8. The peptide according to claim 1, where X ₂₄ represents A. 9. The peptide according to claim 1, where X ₂₄ represents E. 10. The peptide according to claim 1, where X ₂₅ represents R. 11. The peptide according to claim 1, where X ₂₆ represents E. 12. The peptide with the amino acid sequence of SEQ ID NO: 121. 13. A pharmaceutical composition having a corticotropin releasing factor 2 receptor agonist activity, comprising: (a) a safe and effective amount of the peptide according to claim 1; and (b) a pharmaceutically acceptable carrier. 14. A pharmaceutical composition having corticotropin releasing factor 2 receptor agonist activity, comprising: (a) a safe and effective amount of the peptide according to item 12; and (b) a pharmaceutically acceptable carrier. 15. The use of the peptide according to any one of claims 1 to 12 for the manufacture of a medicament for increasing the mass or function of skeletal muscles in a subject in which such an increase is desirable, including (a) identifying a subject in which it is desirable to increase the mass or function of skeletal muscle; and (b) administering to the subject a safe and effective amount of a CRF ₂ R agonist. 16. The use of the peptide according to clause 15, where the subject, which is desirable such an increase, is characterized by the presence of muscular dystrophy or muscle atony. 17. A nucleic acid encoding a peptide with CRF ₂ R agonist activity and characterized by a nucleotide sequence as defined by the amino acid sequence of the peptide according to claim 1. 18. An antibody specific for a peptide with CRF ₂ R agonist activity and which is prepared using the peptide of claim 1. 19. A kit for the prevention or treatment of a disease mediated by CRF ₂ R, containing (a) the peptide according to any one of the preceding paragraphs of the dosage form; (b) instructions for use.