GHRH antagonists for use in method of treating sarcoidosis

Abstract

To provide a pharmaceutical composition for treating lung inflammation in a mammalian subject having a granulomatous pulmonary disease.SOLUTION: A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a GHRH antagonist.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This invention was made with U.S. government support under a Distinguished Scientist grant awarded by the U.S. Department of Veterans Affairs and grant number P30CA240139 awarded by the National Institutes of Health (NIH). The U.S. government has certain rights in this invention.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS AND INCORPORATION-BY-REFERENCE OF ELECTRONICALLY SUBMITTED MATERIALS This application hereby claims priority to U.S. Provisional Patent Application No. 62 / 875,703, filed July 18, 2019, which is incorporated by reference in its entirety.

[0003] The computer readable nucleotide / amino acid sequence listings, filed concurrently herewith and identified as follows, are incorporated by reference in their entirety: 54402A_Seqlisting.txt; Size: 10,859 bytes; Created: July 17, 2020.

[0004] Field of Disclosure The present invention relates to materials and methods for treating sarcoidosis. [Background technology]

[0005] Sarcoidosis is a multisystem granulomatous disease of unknown etiology that is associated with significant morbidity and mortality in the United States and affects hundreds of thousands of people worldwide (see Non-Patent Document 1). Although the etiology of this disease is unknown, there are striking similarities between sarcoidosis and other granuloma-forming disorders, including granulomas induced by mycobacterial and other microbial infections and environmental agents (see Non-Patent Document 2). Sarcoidosis elicits an early inflammatory response in affected organs characterized by the recruitment of Th1 helper cells, followed by the late recruitment of macrophages, leading to granuloma formation. In certain patients, anti-inflammatory responses, including cytokines and apoptosis, are activated to facilitate tissue healing and repair (see Non-Patent Document 3). Approximately 50% of sarcoidosis patients require systemic steroid therapy. In up to 20% of patients, the inflammatory process continues despite steroid treatment, leading to tissue remodeling, including fibrosis (persistent scarring of affected tissues) (see Non-Patent Document 4).

[0006] Given the multiorgan involvement of sarcoidosis in over 50% of patients, treatment of this disease is challenging. Corticosteroids are the cornerstone of treatment, and the U.S. Food and Drug Administration (FDA) has approved only two drug treatments for sarcoidosis (prednisone and Acthar-Gel) (see Non-Patent Documents 5 and 6). However, these drugs induce significant side effects after prolonged use, making them undesirable for long-term treatment. In patients with persistent signs and complex symptoms involving vital organs, treatment must be initiated immediately and continued for several months; therefore, alternative, less toxic, and more tolerable strategies are needed. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Mirsaeidi et al.,Chest.2015;147(2):438-449 [Non-patent document 2] Chen et al.,Clinics in chest medicine.2008;29(3):365-377,vii [Non-patent document 3] Koh et al.,Expert Rev Mol Med.2011;13:e23 [Non-patent document 4] Patterson et al.,Annals of the American Thoracic Society.2013;10(4):362-370 [Non-patent document 5] Miller et al., Ann Intern Med.1952;37(4):776-784 [Non-patent document 6] Baughman et al.,Respir Med.2016;110:66-72 Summary of the Invention

[0008] 1. The present disclosure provides a method of treating sarcoidosis, comprising administering a GHRH antagonist to a mammalian subject in need thereof. The present disclosure further provides the use of a GHRH antagonist for treating sarcoidosis or in the preparation of a medicament for treating sarcoidosis. The present disclosure also provides a GHRH antagonist for use in treating sarcoidosis.

[0009] 2. In various embodiments of the method or use of paragraph 1, the GHRH antagonist has the amino acid sequence (Formula I): 1 -Tyr 1 -D-Arg 2 -Asp 3 -A 4 -Ile 5 -A 6 -Thr 7 -A 8 -Har 9 -A 10 -A 11 -A 12 -Val 13 -Leu 14 -A 15 -Gln16 -A 17 -Ser 18 -Ala 19 -A 20 -A 21 -Leu 22 -Leu 23 -Gln 24 -Asp 25 -Ile 26 -Nle 27 -D-Arg 28 -A 29 -R 2 -R 3 -NH2 (SEQ ID NO:2), wherein R 1 is PhAc (phenylacetyl), Nac (naphthylacetyl), Oct (octanoyl), N-Me-Aib (N-methyl-alpha-aminoisobutyroyl), Dca (dichloroacetyl), Ac-Ada (acetyl-12-aminododecanoyl), Fer (ferulyl), Ac-Amc (acetyl-8-aminocaprylyl), Me-NH-Sub (methyl-NH-suberyl), PhAc-Ada (phenylacetyl 12-aminododecanoyl), Ac-Ada-D-Phe, Ac-Ada-Phe, Dca-Ada (dichloroacetyl-12-aminododecanoyl), Nac (naphthylacetyl), Nac-Ada, Ada-Ada, or CH3(CH2) 10 -CO-Ada; A 4 is Ala or Me-Ala; A 6 is Cpa (para-chlorophenylalanine) or Phe(F)5 (pentafluoro-phenylalanine, also called Fpa5); A 8 is Ala, Pal (pyridylalanine), Dip ((3,3-diphenyl)alanine), or Me-Ala; A 10 is Fpa5, Tyr(Alk), where Alk is Me or Et; A 11 is His or Arg; A 12is Lys, Lys(0-11) (Lys(A0-Al-A2-A3-A4-A5-A6-A7-A8-A9-A10-A11-), Lys(Me)2, or Ornithine; A 15 is Abu (alpha-aminobutyric acid) or Orn; A 17 is Leu or Glu; A 20 is Har (homoarginine) or His; A 21 is Lys, Lys(Me)2 or Orn; A 29 is Har, Arg or Agm (agmatine); R 2 is β-Ala, Amc (8-aminocaprylyl), Apa (5-aminopentanoyl), Ada (12-aminododecanoyl), AE2A (8-amino-3,6-dioxaoctanoyl), AE4P (15-amino-4,7,10,13-tetraoxapentadecanoyl), ε-Lys(α-NH2) (a Lys residue in which the 8-amino group is acetylated by the carbonyl group of the amino acid located at the N-terminus, and the α-amino group of the Lys residue is free), Agm (agmatine), or absent; R 3 is Lys(Oct), Ahx(6-aminohexanoyl), or absent.

[0010] 3. In various forms of the method or use of paragraph 1, the GHRH antagonist is MIA-602, MIA-604, MIA-606, MIA-610, MIA-640, or MIA-690.

[0011] 4. In various forms of the method or use of paragraph 1, the GHRH antagonist is MIA-602.

[0012] 5. In various forms, such as the method or use of any one of paragraphs 1-4, the GHRH antagonist is administered via intradermal, intramuscular, intraperitoneal, intravenous, intraarterial, subcutaneous, epidural, sublingual, intranasal, intracerebral, intracerebroventricular, intrathecal, intravaginal, transdermal, rectal, inhalation, intrapulmonary, intratracheal, intrabronchial, intratracheal, or topical delivery.

[0013] 6. In various forms, such as the method or use of paragraph 5, the GHRH antagonist is administered subcutaneously.

[0014] 7. In various forms, such as the method or use of any one of paragraphs 1-5, the GHRH antagonist is administered via intranasal, inhaled, intrapulmonary, intratracheal, intrabronchial, or intratracheal delivery.

[0015] 8. In various forms of the method or use of any one of paragraphs 1 to 7, the sarcoidosis is pulmonary sarcoidosis. [Brief explanation of the drawings]

[0016] [Figure 1] Figures 1A-1E: Bar graphs showing the percent pulmonary inflammation in mice with sarcoidosis treated with saline, α-melanocortin-stimulating hormone (α-MSH, a melanocortin receptor agonist), MIA-602, and Solu-Medrol (methylprednisolone; a currently FDA-approved drug treatment for sarcoidosis). Pulmonary inflammation was determined and scored by a pulmonary pathologist using hematoxylin and eosin (H&E) staining (Figure 1A), CD68 levels (Figure 1B), PD-1 levels (Figure 1C), PD-L1 levels (Figure 1D), and CD30 levels (Figure 1E). [Figure 2]Figures 2A-2T: The anti-inflammatory activity of MIA-602 in sarcoid-like granulomas from human cells was tested in peripheral blood mononuclear cells (PBMCs) isolated from patients with confirmed sarcoidosis. PBMCs were challenged with microparticles to generate ex vivo granulomas. Figures 2A-2T are bar graphs showing cytokine production (x-axis, pg / ml) in granuloma samples treated with MIA-602 or Solu-Medrol (methylprednisolone) compared to PBMCs not challenged with microparticles and saline-treated granulomas. [Figure 3] Figures 3A-3D: Bar graphs showing the percentage levels of survivin (Figure 3A), Mcl-1 / Bak dimer (Figure 3B), Bcl-xL / Baak dimer (Figure 3C), and active caspase-3 (Figure 3D) in in vitro granuloma samples after treatment with MIA-602, methylprednisolone, or control. [Figure 4] Bar graph showing the % GHRHR immunofluorescence staining in the lungs of mice with sarcoid-like granulomas that developed upon challenge with microparticles compared to saline or control-treated granulomas. [Figure 5] Figures 5A-5E: Bar graphs showing H&E (Figure 5A) and immunohistochemical (IHC) changes of CD30 (Figure 5B), CD68 (Figure 5C), PD-1 (Figure 5D), and PD-L1 (Figure 5E) in the lungs of control mice, saline-treated mice with granulomas, and MIA-602-treated mice with granulomas. Number of mice: 3 control, 4 granulomas, 3 MIA602. [Figure 6] Figures 6A-6C: Bar graphs showing the percent increase in CD45+CD68+, CD45+CD68+PD-1, or CD45+CD68+PD-L1 cells in granulomas from control, MIA-602-treated, and methylprednisolone-treated mice. The percent increase in CD45+CD68+PD-L1 cells was higher after MIA-602 treatment compared to other cell types. [Figure 7]Bar graph showing % NOS2 staining in the lungs of control mice (no granulomas), mice challenged with microparticles that developed granulomas, and mice with granulomas that were treated with MIA-602. [Figure 8] Bar graph showing % nitrotyrosine staining in the lungs of control mice (no granulomas), mice challenged with microparticles that developed granulomas, and mice with granulomas and treated with MIA-602 (long of mouse). [Figure 9] A list of genes that exhibited greater than 2.5-fold differential expression upon treatment of sarcoidosis mice with MIA-602. This list includes the gene's stable identifier (Ensembl database reference number), gene name, and RNA transcript type. DETAILED DESCRIPTION OF THE INVENTION

[0017] The present disclosure provides a method for treating sarcoidosis (e.g., pulmonary sarcoidosis). The method comprises administering a GHRH antagonist to a mammalian subject in need thereof. The data presented herein demonstrate that a GHRH antagonist (e.g., MIA-602) significantly reduces inflammation in an in vivo model of sarcoidosis.

[0018] The term "subject" includes, but is not limited to, humans and non-human mammals, such as wild, domestic, and livestock animals. Preferably, the subject is a human. The subject may be afflicted with any form of sarcoidosis (i.e., sarcoidosis of any organ, such as the lungs).

[0019] Growth hormone-releasing hormone (GHRH) is secreted by the hypothalamus and acts on the pituitary gland to stimulate growth hormone (GH) release. Nearly 2,000 synthetic antagonist analogs of GHRH have been generated by amino acid substitutions at the biologically active N-terminus of GHRH(1-29). Schally et al., Nat. Clin. Pract. Endocrinol. Metab. 4 (1), 33-43 (2008); Zarandi et al., PNAS 91 (25), 12298-302 (1994); Zarandi et al., Peptides 89, 60-70 (2017). The pituitary GHRH receptor (pGHRH-R) is a G protein-coupled, seven-transmembrane domain receptor. Rekasi et al., PNAS 97(19), 10561-6 (2000); Havt et al., PNAS 102(48), 17424-9 (2005). pGHRH-R and its truncated splice variants (SV) are expressed in various human tissues. SV1 differs from pGHRH-R in the amino-terminal extracellular domain. Rekasi, supra.

[0020] In various forms, the GHRH antagonist is a peptide. Various modifications of the GHRH peptide confer antagonist properties. A GHRH fragment comprising residues 1-29, or GHRH(1-29), is the minimal sequence required for biological activity in the pituitary gland. This fragment retains more than 50% of the potency of native GHRH. Many synthetic analogs of GHRH based on the structure of the hGH-RH(1-29)NH2 peptide have been prepared and are contemplated for use in connection with this method herein. hGHRH(1-29)NH2 has the following amino acid sequence: Tyr-Ala-Asp-Ala-Ile 5 -Phe-Thr-Asn-Ser-Tyr 10 -Arg-Lys-Val-Leu-Gly 15 -Gln-Leu-Ser-Ala-Arg 20 -Lys-Leu-Leu-Gln-Asp 25 -Ile-Met-Ser-Arg 29-NH2 (SEQ ID NO:1). GHRH antagonists may include GHRH peptide sequences in which amino acid deletions, insertions, and / or substitutions have been made. GHRH antagonists may also be fragments or modified fragments of GHRH that have the ability to bind to the GHRH receptor and inhibit growth hormone release. These antagonist properties are believed to result from the substitution of various amino acids at the N-terminus of GHRH(1-29)NH2 and acylation with aromatic or nonpolar acids.

[0021] Optionally, the GHRH antagonist is an antagonist described in U.S. Patent Application Publication No. 20150166617 or U.S. Patent No. 8,691,942 (incorporated herein by reference in their entirety and particularly with respect to their description of GHRH antagonists). For example, in various embodiments, the GHRH antagonist is an antagonist having the amino acid sequence (Formula I / SEQ ID NO:2)R 1 -Tyr 1 -D-Arg 2 -Asp 3 -A 4 -Ile 5 -A 6 -Thr 7 -A 8 -Har 9 -A 10 -A 11 -A 12 -Val 13 -Leu 14 -A 15 -Gln 16 -A 17 -Ser 18 -Ala 19 -A 20 -A 21 -Leu 22 -Leu 23 -Gln 24 -Asp 25 -Ile 26 -Nle 27 -D-Arg 28 -A 29 -R 2 -R 3 -NH2, where R 1is PhAc (phenylacetyl), Nac (naphthylacetyl), Oct (octanoyl), N-Me-Aib (N-methyl-alpha-aminoisobutyroyl), Dca (dichloroacetyl), Ac-Ada (acetyl-12-aminododecanoyl), Fer (ferulyl), Ac-Amc (acetyl-8-aminocaprylyl), Me-NH-Sub (methyl-NH-suberyl), PhAc-Ada (phenylacetyl 12-aminododecanoyl), Ac-Ada-D-Phe, Ac-Ada-Phe, Dca-Ada (dichloroacetyl-12-aminododecanoyl), Nac (naphthylacetyl), Nac-Ada, Ada-Ada, or CH3 (CH2) 10 -CO-Ada;A 4 is Ala or Me-Ala; A 6 is Cpa (para-chlorophenylalanine) or Phe(F)5 (also known as Fpa5); A 8 is Ala, Pal (pyridylalanine), Dip ((3,3-diphenyl)alanine), or Me-Ala; A 10 is Fpa5, Tyr(Alk), where Alk is Me or Et; 11 is His or Arg; A 12 Lys, Lys(0-11) (otherwise A 12 is written as a string of lysine residues at positions A, i.e., Lys(A0-Al-A2-A3-A4-A5-A6-A7-A8-A9-A10-A11-), where each A is a lysine), Lys(Me)2, or Orn (ornithine); 15 is Abu (alpha-aminobutyric acid) or Orn; A 17 is Leu or Glu; A 20 is Har (homoarginine) or His; A 21 is Lys, Lys(Me)2 or Orn; A 29is Har, Arg, or Agm (agmatine); R2 is β-Ala, Amc (8-aminocaprylyl), Apa (5-aminopentanoyl), Ada (12-aminododecanoyl), AE2A (8-amino-3,6-dioxaoctanoyl), AE4P (15-amino-4,7,10,13-tetraoxapentadecanoyl), ε-Lys(α-NH2) (a Lys group in which the 8-amino acid is acetylated by the carbonyl group of the amino acid located at the N-terminus, and the α-amino group of the Lys residue is free), Agm (agmatine), or absent; R 3 is Lys(Oct), Ahx(6-aminohexanoyl), or absent.

[0022] Optionally, the GHRH antagonist is MIA-602 [PhAc-Ada], further described in U.S. Patent Application Publication No. 20150166617 (herein incorporated by reference for its discussion of the structure, activity and methods of making MIA-602, MIA-604, MIA-606, MIA-610, MIA-640, and MIA-690). 0 -Tyr 1 , D-Arg 2 , Fpa5 6 , Ala 8 , Har 9 , Tyr(Me) 10 , His 11 , Orn 12 , Abu 15 , His 20 , Orn 21 , Nle 27 , D-Arg 28 , Har 29 ]hGH-RH(1-29)NH2 (SEQ ID NO:8). Alternative GHRH antagonists include Phac-Ada-Tyr 1 -D-Arg 2 -Asp 3 -Ala 4 -Ile 5 -Phe(F)5 6 -Thr 7 -Ala 8 -Har 9 -Tyr(Me)10 -His 11 -Orn 12 -Val 13 -Leu 14 -Abu 15 -Gln 16 -Leu 17 -Ser 18 -Ala 19 -His 20 -Orn 21 -Leu 22 -Leu 23 -Gln 24 -Asp 25 -Ile 26 -Nle 27 -D-Arg 28 -Har 29 -Agm-NH2(MIA-604 / SEQ ID NO:3);Phac-Ada-Tyr 1 -D-Arg 2 -Asp 3 -Ala 4 -Ile 5 -Phe(F)5 6 -Thr 7 -Me-Ala 8 -Har 9 -Tyr(Me) 10 -His 11 -Orn 12 -Val 13 -Leu 14 -Abu 15 -Gln 16 -Leu 17 -Ser 18 -Ala 19 -His 20 -Orn 21 -Leu 22 -Leu 23 -Gln 24 -Asp 25 -Ile 26 -Nle 27 -D-Arg 28 -Har 29 -Agm-NH2(MIA-606 / SEQ ID NO:4);Phac-Tyr 1 -D-Arg 2 -Asp 3 -Ala 4 -Ile 5-Cpa 6 -Thr 7 -Ala 8 -Har 9 -Fpa5 10 -His 11 -Orn 12 -Val 13 -Leu 14 -Abu 15 -Gln 16 -Leu 17 -Ser 18 -Ala 19 -His 20 -Orn 21 -Leu 22 -Leu 23 -Gln 24 -Asp 25 -Ile 26 -Nle 27 -D-Arg 28 -Har 29 -Ada-NH2(MIA-610 / SEQ ID NO:5);Phac-Ada-Tyr 1 -D-Arg 2 -Asp 3 -Ala 4 -Ile 5 -Cpa 6 -Thr 7 -Ala 8 -Har 9 -Fpa5 10 -His 11 -Orn 12 -Val 13 -Leu 14 -Abu 15 -Gln 16 -Glu 17 -Ser 18 -Ala 19 -His 20 -Orn 21 -Leu 22 -Leu 23 -Gln 24 -Asp 25 -Ile 26 -Nle 27 -D-Arg 28 -Har 29 -Ada-NH2(MIA-640 / SEQ ID NO:6);Phac-Ada-Tyr 1-D-Arg 2 -Asp 3 -Ala 4 -Ile 5 -Cpa 6 -Thr 7 -Ala 8 -Har 9 -Fpa5 10 -His 11 -Orn 12 -Val 13 -Leu 14 -Abu 15 -Gln 16 -Leu 17 -Ser 18 -Ala 19 -His 20 -Orn 21 -Leu 22 -Leu 23 -Gln 24 -Asp 25 -Ile 26 -Nle 27 -D-Arg 28 -Har 29 -NH2 (MIA-690 / SEQ ID NO:7). The amino acid sequences of the above peptides are numbered corresponding to the amino acid residues of hGHRH(1-29) (SEQ ID NO:1).

[0023] The present disclosure provides methods of treating sarcoidosis in a subject in need thereof. "Treating" sarcoidosis does not require 100% relief. Any reduction in sarcoidosis or symptoms of sarcoidosis (e.g., inflammation, granuloma formation, granuloma size) in improving quality of life constitutes a beneficial biological effect in the subject. The progress of this method in treating sarcoidosis may be confirmed using any suitable method, such as biomarker detection / measurement in biological (e.g., blood) samples, chest imaging (e.g., CT scan), and PET-CT scan. In certain embodiments, the method provides at least a 50%, at least a 65%, at least a 75%, at least a 80%, at least a 85%, at least a 90%, at least a 95%, or at least a 99% reduction or improvement in a disease indicator, parameter, or symptom, such as a reduction in angiotensin-converting enzyme (ACE), SIL2R, or CRP biomarkers, compared to prior treatment, or at least a 50% reduction in a disease indicator, parameter, or symptom compared to that achieved by treatment with prednisone (administered prior to the method, or in a suitable patient). In various embodiments, "treatment" also encompasses stabilization of the disease, i.e., controlled or no further progression of the disease (e.g., granuloma burden not increasing within a given time frame, or increasing by less than 10%, preferably less than 5%).

[0024] Alternatively or additionally, the treatments described herein may improve the stage of the disease or reduce the severity within a stage. Commonly used stages for sarcoidosis include Stage I, where granulomas are primarily present in the lymph nodes; Stage II, where granulomas are present in the lungs and lymph nodes; Stage III, where granulomas are primarily present in the lungs and lymph nodes are shrinking; and Stage IV, where pulmonary fibrosis is present.

[0025] Disease progression in sarcoidosis is determined using any of a variety of clinical techniques, such as biopsy of the affected organ(s) to identify granulomas, blood tests, bronchoscopy, x-rays, neurological tests (e.g., electromyography, evoked potentials, spinal tap, or nerve conduction tests), high-resolution computed tomography (CT) scans, magnetic resonance imaging (MRI), positron emission tomography (PET) scans, pulmonary function tests, and ultrasound.

[0026] The individual dosing regimen for each individual subject will depend, in part, on the amount of antagonist administered, the route of administration, and the cause and extent of any side effects. The amount administered to a subject (e.g., a human) in accordance with the present disclosure should be sufficient to affect the desired response (i.e., to improve, prevent, or ameliorate the patient's unwanted illness, disease, or symptoms) in a reasonable time frame. A therapeutically effective amount of a GHRH antagonist is typically an amount sufficient to achieve an effective systemic concentration or local concentration in target tissues when the GHRH antagonist is administered in a physiologically tolerable excipient composition.

[0027] The dose of the GHRH antagonist is optionally about 0.005 mg / kg to about 100 mg / kg. In various embodiments, the GHRH antagonist is administered at a dose of about 0.05 mg / kg to about 20 mg / kg. In some embodiments, the GHRH antagonist is administered at a dose of about 0.01 mg / kg / dose to about 50 mg / kg / dose, about 0.01 mg / kg / dose to about 25 mg / kg / dose, about 0.1 mg / kg to about 15 mg / kg, or about 1 mg / kg to about 10 mg / kg. Optionally, the dose is given once daily or divided into 2 to 4 administrations per day. When the GHRH antagonist is administered intravenously to a human patient, the dose is optionally divided into 1 to 4 bolus injections per day or given as a continuous infusion.

[0028] The GHRH antagonist may be administered daily, at least once a week, at least twice a week, at least three times a week, at least four times a week, at least five times a week, six times a week, every two weeks, every three weeks, every four weeks, every five weeks, or every six weeks. The duration of treatment (with multiple administrations of the antagonist) will depend on the nature and severity of the disease, as well as the presence of any side effects. Examples of treatment durations include, but are not limited to, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 9 months, and 12 months.

[0029] Administration methods can include, but are not limited to, oral administration and parenteral administration, including, but not limited to, intradermal, intramuscular, intraperitoneal, intravenous, intraarterial, subcutaneous, epidural, sublingual, intranasal, intracerebral, intraventricular, intrathecal, intravaginal, transdermal, rectal, inhalation, intrapulmonary, intratracheal, intrabronchial, intratracheal, or topical (e.g., ear, nose, eye, or skin) delivery. The antagonist is administered subcutaneously in various forms. In other forms, such as when the subject suffers from pulmonary sarcoidosis, the antagonist is administered via intranasal, inhalation, intrapulmonary, intratracheal, intrabronchial, or intratracheal delivery.

[0030] Optionally, the GHRH antagonist is administered alone or in combination with other medications (simultaneously or sequentially), optionally as a single combined formulation or as separate compositions. In some forms, the method includes administering multiple GHRH antagonists. Alternatively or additionally, the GHRH antagonist is optionally administered in combination with other anti-inflammatory agents, such as steroids. Alternatively or additionally, the GHRH antagonist is optionally administered in combination with one or more disease-modifying antirheumatic drugs (DMARDs, e.g., methotrexate, azathioprine, or leflunomide), monoclonal antibodies (e.g., infliximab, adalimumab, rituximab, or golimumab), colchicine, hormone therapy (e.g., corticotropin), antibiotics, and / or pentoxifylline.

[0031] GHRH antagonists may be administered in the form of pharmaceutically acceptable non-toxic salts, such as acid addition salts.Examples of such acid addition salts include hydrochloride, hydrobromide, sulfate, phosphate, fumarate, gluconate, tannate, maleate, acetate, trifluoroacetate, citrate, benzoate, succinate, alginate, pamoate, malate, ascorbate, tartrate, and the like.Particularly preferred antagonists are salts with low solubility, such as pamoate and the like.

[0032] Formulations containing a GHRH antagonist and a suitable carrier may be in solid dosage forms, including, but not limited to, softgels, tablets, capsules, cachets, pellets, pills, powders, and granules; topical dosage forms, including, but not limited to, solutions, powders, fluid emulsions, fluid suspensions, semisolids, ointments, pastes, creams, gels, and jellies; and parenteral dosage forms, including, but not limited to, solutions, suspensions, emulsions, and powders. In some embodiments, a single dose may comprise one or multiple administrations (i.e., multiple injections or multiple pills to arrive at a single dose / amount of antagonist).

[0033] The GHRH antagonist may be contained in a formulation together with pharmaceutically acceptable diluents, fillers, disintegrants, binders, lubricants, surfactants, hydrophobic vehicles, water-soluble vehicles, emulsifiers, buffers, wetting agents, humectants, solubilizers, preservatives, and the like. Means and methods of administration are known in the art, and those skilled in the art can refer to various pharmacological reference materials for guidance. For example, Modern Pharmaceutics, Banker & Rhodes, Marcel Dekker, Inc. (1979); and Goodman & Gilman's The Pharmaceutical Basis of Therapeutics, 6th Edition, MacMillan Publishing Co., New York (1980). The pharmaceutical composition may include a suitable solid or gel phase carrier or excipient. Examples of such carriers or excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycol.

[0034] The invention thus generally described will be more readily understood by reference to the following examples, which are offered by way of illustration and not by way of limitation. [Example]

[0035] General method Preparation of MIA-602 The chemical structure of MIA-602 is [PhAc-Ada 0 , D-Arg 2 , Fpa5 6 , Ala 8 , Har 9 , Tyr(Me) 10 , His 11 , Orn 12 , Abu 15 , His 20 , Orn 21 , Nle 27 , D-Arg 28 , Har 29]hGH-RH(l-29)NH2. This compound was dissolved in 100% dimethyl sulfoxide (DMSO, ACS grade, Sigma) for storage and diluted 1:1000 with the corresponding medium to a final concentration of 1 μM. In vitro and in vivo control groups received a placebo with the same volume and concentration of DMSO.

[0036] Development of microparticles Microparticles were produced as previously described (Zhang et al., Sci Rep 2020;10:7277). Microparticles were generated by sonicating and heating live bacilli from rough colonies of a clinical strain of Mycobacterium abscessus (MAB). High-quality images of non-infectious MAB particles were obtained by scanning electron microscopy (SEM).

[0037] human blood samples Blood samples were collected from nine randomly selected patients with confirmed pulmonary sarcoidosis at the University of Miami Sarcoidosis Biobanking, who were subsequently matched for age, sex, and race with healthy controls. To avoid the inconvenience and risks associated with additional blood collection procedures, 10 ml blood specimens were collected. Patients with an Hgb <7 mg / dL at that time were excluded from participation in the study.

[0038] Mature in vitro granuloma-like formation In vitro granulomas were developed by challenging PBMCs with microparticles as previously described (Zhang et al, Sci Rep 2020;10:7277).

[0039] Mouse model exposure to MAB microparticles Granulomatous responses in mouse lungs were developed as previously described (Zhang et al, Front Immunol 2019;10:2888).

[0040] ELISA (Zhang et al., Sci Rep 2020;10:7277) PBMCs were lysed in lysis buffer (Cell Signaling Technology, Beverly, MA) with a protease inhibitor cocktail (Cell Signaling Technology, Beverly, MA) and sonicated for three 2-second bursts with at least 1 minute on ice between each 2-second pulse. Samples were centrifuged at 10,000 x g and 4°C for 5 minutes, and the supernatant was collected. Protein concentration was determined using Cell Signaling Technology's BCA protein assay kit. This methodology is further described in Zhang et al., Sci Rep 2020;10:7277.

[0041] Thirty micrograms of total protein was mixed with reducing sample buffer and used in the mitochondrial apoptosis assay, which was performed using a Bio-Rad kit (171-WAR3CK) according to the kit's instructions.

[0042] To measure cytokines in the medium, aliquots of the supernatant were analyzed, thawed, and spun at 12,000 rpm for 10 minutes to separate any particulate material at the bottom. Fifty microliters of undiluted medium from each sample was seeded into a 96-well V-bottom plate by manual pipetting according to a predefined protocol. The aliquots were packaged in Parafilm and kept at 4°C in a humidified chamber for no longer than 72 hours. Growth factors and their receptor capture antibodies were reconstituted and diluted according to the manufacturer's specifications, and 50 μl was seeded into each well of each high-binding 96-well half-area plate, which was then sealed and incubated overnight at 4°C. Cytokine levels were measured using Invitrogen's Procartaplex human TH1 / TH2 cytokine panel 11plex (epx110-10810-901).

[0043] Immunofluorescence confocal microscopy Details of the methodology used for confocal microscopy are discussed in Zhang et al., Front Immunol 2019;10:2888. Briefly, mice were sacrificed on day 14, and the left lungs were harvested. Lungs were filled with 10% buffered formalin and formalin-fixed for at least 72 hours before IHC staining. H&E staining was used to determine inflammatory pathology.

[0044] For immunofluorescence, paraffin-embedded serial sections (5 μm) were first subjected to standard deparaffinization and rehydration procedures. Sections were then probed with GHRHR (Origene, cat# TA311715) as the primary antibody and Sigma anti-rabbit antibody (cat# F-9887) as the secondary antibody. Nuclei were counterstained with DAPI. All reagents were from Sigma-Aldrich. Tissue sections were analyzed using fluorescence microscopy and ImageJ software (version 6.0; NIH) to quantify fluorescence intensity. In Trichrome-stained slides, blue staining (collagen content) was also quantitatively analyzed using ImageJ.

[0045] Confocal immunofluorescence images were acquired at the University of Miami McKnight Analytical Imaging Core Facility using a Leica DM6000 microscope equipped with an SP5 confocal module. Captured images were processed using Velocity Software version 6.1.1 (Perkin-Elmer, Waltham, MA).

[0046] For immunohistochemistry, 5 μm paraffin sections were processed by deparaffinization and rehydration, followed by endogenous peroxidase blocking (1% H2O2 in methanol for 20 min) and antigen retrieval (boiling in 10 mM citrate buffer for 30 min). Tissue sections were blocked with 2% goat or horse serum (Vector Laboratories) and incubated overnight at 4°C with antibodies to CD68 (Proteintech, Cat#25747-1-AP), PD-1 (Cell Signaling, Cat#84651), PD-L1 (Proteintech, Cat#17952-1-AP), CD30 (Lsbio, Cat#LS-cl62069), CD3 (Cell Signaling, Cat#99940), iNOS (Invitrogen, Cat#PAI-036), or nitrotyrosine (Novus, NBP2-54606). After washing five times with TBST, sections were then exposed to secondary antibodies (Vector Laboratories, Cat#PI-2000). Immunoreactivity was detected using the ABC Elite kit (Vector Laboratories). DAB was used as the final chromogen and hematoxylin was used as the nuclear counterstain. Negative controls for all antibodies were made by replacing the primary antibody with non-immunogenic IgG.

[0047] Pulmonary inflammation was scored using three fields of maximum infiltrate intensity at 100X magnification as previously described (Zhang et al., Front Immunol 2019;10:2888). The area of ​​inflammation was measured and averaged across the three active fields examined.

[0048] RNA isolation and analysis RNA from mouse lungs was extracted using the RNA Miniprep Plus kit (Zymo Research). Briefly, whole lungs were homogenized in TRI Reagent, and total RNA extraction was performed according to the manufacturer's instructions, with additional DNase treatment. Sample quantity and quality were determined using a NanoDrop spectrophotometer and an Agilent Bioanalyzer 2100, respectively (Zhang et al., Front Immunol 2019;10:2888).

[0049] RNA library preparation and sequencing were performed. Briefly, the quantity and quality of total RNA were determined using an Agilent Bioanalyzer. At least 300 ng of total RNA was used as input for the KAPA RNA HyperPrep Kit with RiboErase according to the manufacturer's protocol to generate ribosomal RNA-depleted sequencing libraries. Sequencing was performed on an Illumina NextSeq 500, generating approximately 40 million single-end 75-bp reads per sample. Sequencing data were processed through a bioinformatics pipeline, including quality control, alignment to the hg19 human reference genome, and gene quantification. Count data were input into edgeR software for differential expression analysis. Counts were normalized using the trimmed mean M (TMM) method, and a general linear model with the sample as a blocking factor was used to account for compositional differences between libraries and differential expression pair analysis. Genes were considered statistically different if the false discovery rate p-value (FDR) was ≤0.05.

[0050] Flow cytometry Mice were sacrificed on day 14, and the left lungs were collected for pathological examination after perfusion of the right ventricle with 10 ml of PBS.

[0051] The upper half of the left lung tissue (excluding the trachea, main bronchi, and carina) was removed and rinsed with PBS to flush out blood. The tissue was crushed and dispersed with scissors to increase the total surface area. To generate a single-cell suspension, the rubber end of a 5 ml plastic syringe was used to sieve the cells through a 100 μm cell strainer, with successive rinses of ice-cold RPMI 1640. The cell suspension was again sieved through a 70 μm cell strainer and thoroughly rinsed with 3 ml of DNAse-containing wash buffer followed by 15 ml of DNAse-free wash buffer. The sample was centrifuged at 286 × g and 18°C ​​for 5 minutes, and the supernatant was discarded (Posel et al. J Vis Exp 2016:53658).

[0052] cells (10 6 Cells (100 cells / ml) were resuspended in 100 μl of protein blocking solution containing 5 μl of fluorescently conjugated antibodies: CD8 (Biolegend Cat#100714, CD45 (Biolegend Cat#103130), CD68 (Biolegend Cat#137004), PD-1 (Biolegend Cat#135219), PD-L1 (Biolegend Cat#124308), CD4 (Biolegend Cat#100510), CD11b (Biolegend Cat#101243), CD11c (Biolegend Cat#117318), F4 / 80 (Biolegend Cat#123146), or IFNg (Biolegend Cat#505836). Samples were analyzed on a BD LSR 1000 sbsp buffer using BD FACSDiva software. The data were analyzed using a Fluorescence Intensity Analyzer II flow cytometer, and data analysis was performed using Flowjo software (TreeStar, Ashland, OR). Cell populations were identified using a sequential gating strategy, and expression of activation markers was presented as median fluorescence intensity. Lung immune cells were classified based on FC marker expression as previously described (Misharin et al., Am J Respir Cell Mol Biol 2013;49:503-510).

[0053] Example 1 The following example demonstrates the treatment of sarcoidosis in vivo using the GHRH antagonists of the present disclosure.

[0054] A murine model of pulmonary sarcoidosis was established in C57B1 / 6 mice by intratracheal administration of microparticles. The tongue was removed with a small spatula, and microparticles were inserted into the trachea through a 20G angiographic catheter tube at the laryngeal opening and advanced into the main bronchus until resistance was reached. After tube placement, microparticles were administered, with an initial dose of 5 x 10 M. abscessus in 50 μL. 8 CFU, and the next three doses were 2 × 10 M. abscessus in 20 μL. 8 CFU. The control group received 20 μL of PBS intratracheally only. Mice receiving microparticles developed non-caseating granulomas in the lungs, as observed using H&E staining and immunohistochemical staining for the macrophage marker CD68, CD4, and PD-L1.

[0055] Four groups of mice demonstrated pulmonary sarcoidosis, along with a fifth untreated group that served as a control without sarcoidosis. The first sarcoidosis group received MIA-602 (5 μg / day) administered via intraperitoneal injection. The second sarcoidosis group received α-melanocyte-stimulating hormone (α-MSH), the third sarcoidosis group received steroid treatment (methylprednisolone, the current first-line treatment for sarcoidosis), and the fourth group received saline only. Mice were sacrificed after 2 weeks, and inflammation in lung samples was graded. As shown in Figures 1A–E, the untreated sarcoidosis group exhibited significant inflammation in the lungs. The sarcoidosis group treated with MIA-602 and α-MSH had lower inflammation scores. Steroid treatment did not affect inflammation.

[0056] The above results demonstrate for the first time that a growth hormone-releasing hormone receptor antagonist (herein MIA-602) is effective in treating sarcoidosis.

[0057] Example 2 The following examples demonstrate that peptides of the present disclosure positively affect the cytokine profile of treated granulomas.

[0058] Granulomas and PBMCs were collected from five subjects with sarcoidosis. Samples were divided into the following groups: control (unchallenged), granulomas challenged with 10:1 microparticle treatment and left untreated, challenged granulomas treated with 1 μM MIA-602 in vitro, and challenged granulomas treated with methylprednisolone in vitro. The medium was removed 48 hours after treatment. Cytokines were measured using a multiplex ELISA device.

[0059] There were significant differences in the expression of several cytokines in granulomas compared to PBMCs. MIA-602 significantly reduced the cytokine production of IL2, IL7, IL10, IL12, IL15, IL17A, CCL2, CCL5, IFNα, and CXCL9 in granulomas. See Figures 2A-2T.

[0060] Example 3 The ability of the disclosed peptide MIA-602 to affect respiration and apoptosis in granuloma cells was investigated. The specific steps of apoptosis and mitochondrial dynamics in granulomas have not been fully elucidated. To test whether MIA-602 had pro- or anti-apoptotic effects, protein levels of pro-apoptotic factors (active caspase-3) and anti-apoptotic factors (survivin, Bcl-xL / Bak dimer, and Mcl-1 / Bak dimer) were measured in an in vitro granuloma model. PBMCs from five subjects with confirmed sarcoidosis were divided into the following groups: control (unchallenged), granulomas challenged with 10:1 treatment of microparticles made from M. abscessus cell walls, granulomas treated with 1 μM MIA-602, and methylprednisolone (138 μM). The medium was removed 48 hours after treatment.

[0061] As shown in Figure 3A, survivin levels showed no statistical difference between the groups. Mcl-1 / Bak dimer levels were significantly reduced in untreated granulomas but restored by MIA-602 treatment (Figure 3B). BclxL / Bak dimer levels were increased in MIA-602-treated granulomas compared to saline-treated granulomas (Figure 3C). Active caspase-3 levels were significantly increased in granulomas compared to PBMCs, possibly due to early lymphocyte activation (Zhang C et al., Sci Rep 2020;10:7277). MIA-602 further increased active caspase-3 (Figure 3D). These data suggest that treatment with GHRH antagonists did not increase apoptosis in granulomas.

[0062] Example 4 A murine lung granuloma model was established to examine the type I IFN pathway after exposure to M. abscessus cell wall. This model is applicable to pulmonary sarcoidosis testing due to its characteristics as a non-infectious lung granuloma model. C57B1 / 6 mice were used to develop the model (Zhang et al., Front Immunol 2019;10:2888).

[0063] Mice were treated with 5 μg of the disclosed peptide MIA-602 per day via intraperitoneal injection. Mice were sacrificed two weeks later, and lung inflammation was graded by a pulmonary pathologist. Lung samples were stained with H&E, CD68, PD1, PD-L1, and CD30 and scored based on the percentage of cells expressing each marker.

[0064] As shown in Figure 4, the granuloma group had significant inflammation in the lungs, while mice treated with MIA-602 had lower inflammation scores. Figure 4 shows quantification of GHRHR immunofluorescence staining after MIA-602 treatment in the granulomatous reaction of mouse lungs.

[0065] Lung inflammation scores were significantly higher in the granuloma group. Lung inflammation was nearly normalized in mice treated with MIA-602, as shown in Figures 5A-5E. CD30 cells were statistically significantly increased in lungs with granulomas and non-significantly decreased in MIA-602-treated mice. The percentage of CD68+ cells in the lungs increased in subjects suffering from granulomas and decreased in MIA-602-treated subjects, but the changes were not statistically significant. This pattern was observed for PD-1+ and PD-L1+ cells in the granuloma and MIA-602-treated groups.

[0066] This animal model study determined that GHRHR is significantly increased in sarcoidosis lungs. MIA-602 has anti-inflammatory properties that significantly reduce the number of inflammatory cells in lung tissue.

[0067] Example 5 Flow cytometry demonstrated that CD68+ cells were reduced after MIA-602 therapy in a mouse model of sarcoidosis. Mice were divided into groups: control, challenged with microparticles and treated with saline, challenged and treated with MIA-602 (5 μg), and challenged and treated with methylprednisolone (100 μg). Two weeks later, lungs were collected from all groups, and lung single cells were prepared for flow cytometry analysis. As shown in Figure 6A, the population of CD45+CD68+ cells significantly increased in challenged mice, and this population was significantly reduced by MIA-602 treatment.

[0068] It was hypothesized that MIA-602 would restore the number of CD68+ cells expressing PD-1. Figure 6B shows that the number of CD45+CD68+ cells that expressed PD-1 was significantly reduced in challenged mice with granulomatous responses in the lungs. The percentage of these cells significantly increased after MIA-602 treatment.

[0069] To confirm that granuloma-bearing mouse lungs exhibited a higher percentage of CD45+CD68+ cells expressing PD-L1, single lung cells were stained in all experimental groups. As shown in Figure 6C, the population of CD45+CD68+PD-L1 cells was significantly increased in granulomas, and a higher percentage was detected after MIA-602 treatment. The anti-inflammatory effect of MIA-602 was primarily due to the reduction of CD68+ cells, and PD-1 and PD-L1 play a role in this process.

[0070] Example 6 Inducible nitric oxide synthase (iNOS) produces nitric oxide and plays a crucial role in granuloma development. iNOS is expressed in macrophages after exposure to bacterial lipopolysaccharide and IFNγ (Facchetti et al., Am J Pathol 1999;154:145-152). To understand the effect of MIA-602 on nitric oxide responses, iNOS and nitrotyrosine (as an indicator of NO function) were detected by staining the lungs of mice challenged with microparticles (a sarcoidosis model) and injected daily with saline, treated with daily intraperitoneal injections of MIA-602 (5 μg), and unchallenged controls that did not receive MIA-602. Lung tissues were harvested 3 weeks later, and the tissues were stained for NOS2 and nitrotyrosine. As shown in Figure 7, NOS2 expression was increased in sarcoid-like granulomas and subsequently reduced after MIA-602 treatment. This finding was not statistically significant.

[0071] Nitrotyrosine expression was statistically significantly increased in the lungs of mice with sarcoid-like granulomas, as shown in Figure 8. Treatment with MIA-602 reduced nitrotyrosine, but this reduction was not statistically significant.

[0072] Challenge with microparticles activated iNOS and increased nitrotyrosine in the lungs. MIA-602 reduced both in these experiments, but the reduction was not statistically significant. These experiments suggest an anti-nitrosative effect of MIA-602 in a mouse model of sarcoidosis.

[0073] Example 7 Transcriptomic changes in a mouse model of sarcoidosis were investigated. RNA was extracted from the lungs of mice challenged with microparticles (a sarcoidosis model) and treated with daily saline injections or daily intraperitoneal injections of MIA-602 (5 μg), as well as from the lungs of unchallenged control mice that did not receive MIA-602. RNA was isolated from lung tissue over the course of 3 weeks, and RNA sequencing was performed. Compared to saline-treated granuloma-bearing mice, 778 genes were upregulated and 293 genes were downregulated (508 protein coding or TEC genes and the remaining noncoding genes). Genes that exhibited greater than 2.5-fold differential expression upon MIA-602 treatment are presented in Figure 9. In various embodiments, the present disclosure provides a method of characterizing a subject's response to a sarcoidosis treatment, comprising measuring the expression of one or more of the genes depicted in Figure 9. In various embodiments, a 2.5-fold differential expression compared to pre-treatment expression levels indicates a therapeutic response to the treatment (i.e., the disorder is treated and, alternatively, the symptoms of the disorder are alleviated, etc.).

[0074] This entire document is to be related as a single disclosure, and all combinations of features described herein should be understood to be contemplated, even if the combinations are not found together in the same sentence, paragraph, or section of this document. Additionally, the present invention includes, as additional aspects, all aspects of the invention narrower in scope than the variations specifically mentioned above. With respect to aspects of the invention described or claimed with "a" or "an," these terms should be understood to mean "one or more," unless the context clearly dictates a more restrictive meaning. With respect to elements described as one or more within a set, it should be understood that all combinations within that set are contemplated. When aspects of the invention are described as "comprising" a feature, it is also contemplated that the aspect "consists of" or "consists essentially of" that feature.

[0075] The applicant(s) have invented the full scope of the claims appended hereto, but the claims appended hereto are not intended to include within their scope prior art work by others. Therefore, if statutory prior art within the scope of a claim is brought to the applicant(s)' attention by the Patent Office or other entity or person, the applicant(s) reserve the right to exercise their right of amendment under applicable patent law to redefine the subject matter of such claim to specifically exclude such statutory prior art or obvious variations of the statutory prior art from the scope of such claim. Variations of the invention defined by such amended claims are also intended as aspects of the invention. Additional features and variations of the invention will be apparent to those skilled in the art from the entirety of this application, and all such features are intended as aspects of the invention.

[0076] All publications, patents, and patent applications cited in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those skilled in the art in light of the teachings of the present invention that certain changes and modifications can be made without departing from the spirit or scope of the appended claims.

Claims

1. A method of treating sarcoidosis comprising administering a GHRH antagonist to a mammalian subject in need thereof.

2. The GHRH antagonist has the amino acid sequence (Formula I / SEQ ID NO:2): １ -Tyr １ -D-Arg ２ -Asp ３ -A ４ -Ile ５ -A ６ -Thr ７ -A ８ -Har ９ -A １０ -A １１ -A １２ -Val １３ -Leu １４ -A １５ -Gln １６ -A １７ -Ser １８ -Ala １９ -A ２０ -A ２１ -Leu ２２ -Leu ２３ -Gln ２４ -Asp ２５ -Ile ２６ -Nle ２７ -D-Arg ２８ -A ２９ -R ２ -R ３ -NH ２ where R １ is selected from PhAc (phenylacetyl), Nac (naphthylacetyl), Oct (octanoyl), N-Me-Aib (N-methyl-alpha-aminoisobutyroyl), Dca (dichloroacetyl), Ac-Ada (acetyl-12-aminododecanoyl), Fer (ferulyl), Ac-Amc (acetyl-8-aminocaprylyl), Me-NH-Sub (methyl-NH-suberyl), PhAc-Ada (phenylacetyl 12-aminododecanoyl), Ac-Ada-D-Phe, Ac-Ada-Phe, Dca-Ada (dichloroacetyl-12-aminododecanoyl), Nac (naphthylacetyl), Nac-Ada, Ada-Ada, or CH ３ (CH ２ ) １０ -CO-Ada; A ４ is Ala or Me-Ala; A ６ is Cpa (para-chlorophenylalanine) or Phe (F) ５ and A ８ is Ala, Pal (pyridylalanine), Dip ((3,3-diphenyl)alanine), or Me-Ala; A １０ is Fpa5, Tyr(Alk), where Alk is Me or Et; A １１ is His or Arg; A １２ is Lys, Lys(0-11) (Lys(A0-Al-A2-A3-A4-A5-A6-A7-A8-A9-A10-A11-), Lys(Me) ２ or Orn (ornithine); A １５ is Abu (alpha-aminobutyric acid) or Orn; A １７ is Leu or Glu; A ２０ is Har (homoarginine) or His; A ２１ But, Lys, Lys(Me) ２ Or Orn; A ２９ is Har, Arg or Agm (agmatine); R ２ β-Ala, Amc (8-aminocaprylyl), Apa (5-aminopentanoyl), Ada (12-aminododecanoyl), AE ２ A (8-amino-3,6-dioxaoctanoyl), AE ４ P(15-amino-4,7,10,13-tetraoxapentadecanoyl), ε-Lys(α-NH ２ ) (a Lys residue in which the 8-amino group is acetylated by the carbonyl group of the amino acid located at the N-terminus, and the α-amino group of the Lys residue is free), Agm (agmatine), or absent; R ３ 2. The method of claim 1, wherein is Lys(Oct), Ahx(6-aminohexanoyl), or absent.

3. 2. The method of claim 1, wherein the GHRH antagonist is MIA-602, MIA-604, MIA-606, MIA-610, MIA-640, or MIA-690.

4. 2. The method of claim 1, wherein the GHRH antagonist is MIA-602.

5. 10. The method of claim 1, wherein the GHRH antagonist is administered via intradermal, intramuscular, intraperitoneal, intravenous, intraarterial, subcutaneous, epidural, sublingual, intranasal, intracerebral, intraventricular, intrathecal, intravaginal, transdermal, rectal, inhalation, intrapulmonary, intratracheal, intrabronchial, intratracheal, or topical delivery.

6. 6. The method of claim 5, wherein the GHRH antagonist is administered via intranasal, inhaled, intrapulmonary, intratracheal, intrabronchial, or intratracheal delivery.

7. 4. The method of claim 3, wherein the GHRH antagonist is administered via intradermal, intramuscular, intraperitoneal, intravenous, intraarterial, subcutaneous, epidural, sublingual, intranasal, intracerebral, intraventricular, intrathecal, intravaginal, transdermal, rectal, inhalation, intrapulmonary, intratracheal, intrabronchial, intratracheal, or topical delivery.

8. 8. The method of claim 7, wherein the GHRH antagonist is administered via intranasal, inhaled, intrapulmonary, intratracheal, intrabronchial, or intratracheal delivery.

9. 5. The method of claim 4, wherein the GHRH antagonist is administered via intradermal, intramuscular, intraperitoneal, intravenous, intraarterial, subcutaneous, epidural, sublingual, intranasal, intracerebral, intraventricular, intrathecal, intravaginal, transdermal, rectal, inhalation, intrapulmonary, intratracheal, intrabronchial, intratracheal, or topical delivery.

10. 10. The method of claim 9, wherein the GHRH antagonist is administered via intranasal, inhaled, intrapulmonary, intratracheal, intrabronchial, or intratracheal delivery.

11. 2. The method of claim 1, wherein the sarcoidosis is pulmonary sarcoidosis.

12. 4. The method of claim 3, wherein the sarcoidosis is pulmonary sarcoidosis.

13. 5. The method of claim 4, wherein the sarcoidosis is pulmonary sarcoidosis.

14. 7. The method of claim 6, wherein the sarcoidosis is pulmonary sarcoidosis.

15. 9. The method of claim 8, wherein the sarcoidosis is pulmonary sarcoidosis.

16. 11. The method of claim 10, wherein the sarcoidosis is pulmonary sarcoidosis.

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