Novel synthesized kpsat peptides for chronic lung disease treatment

Abstract

Novel kisspeptin-13 or KP-13 peptides and KP-13 satellites or modified peptides (KPSATs) are disclosed having improved therapeutic benefits in regulating pathophysiological changes in various tissues, particularly lung tissue. These KPSATs have improved antiproliferative effects and non-cytotoxic effects on airway smooth muscle cells and tissue.

Description

NOVEL SYNTHESIZED KPSAT PEPTIDES FOR CHRONIC LUNG DISEASE TREATMENTGovernment Support

[0001] This invention was made with government support under Grant No. R01 HL146705 (FAR031087), awarded by the National Institutes of Health (NIH). The government has certain rights in the invention.Cross-Reference to Related Application(s)

[0002] This application claims priority to U.S. Provisional Application No. 63 / 672,000 filed July 16, 2024 and entitled “NOVEL SYNTHESIZED KPSAT PEPTIDES FOR CHRONIC LUNG DISEASE TREATMENT,” which is hereby incorporated by reference in its entirety.Sequence Listing

[0002] The instant application contains a Sequence Listing that has been submitted in Extensible Markup Language format via EFS-Web and is hereby incorporated by reference in its entirety The xml copy, created on July 15, 2025, is named 97529.2.USU1 Sequence Listing. xml, and is 113,905 bytes in size.Field

[0003] The various embodiments herein relate to novel kisspeptin receptor (KISS1 R) agonists derived from kisspeptin-13 (KP-13) and are termed as KP-13 satellites (KPSATs), which display improved therapeutic benefits in regulating pathophysiological changes in various tissues, particularly lung tissue. These KPSATs have improved antiproliferative and / or anti-remodeling effects without having cytotoxic effects on airway smooth muscle (ASM) cells and tissue.Background

[0004] Kisspeptins are neuropeptide hormones that aid in regulating pubertal development and sex hormone signaling. Recent studies have explored KISS1R signaling outside of neural and sex tissues, with growing data suggesting its involvement in both disease pathology and the maintenance of healthy tissues

[0005] GPR54, also called KISS1R, is a Rhodopsin-like GPCR in the A5 subfamily that interacts with the peptide family of Kisspeptins (Guzman, S., et al. , KISS1 / KISS1 R in Cancer: Friend or Foe? Frontiers in Endocrinology, 2018. 9; Millar, R P. and A.V. Babwah, KISS1R: Hallmarks of an Effective Regulator of the Neuroendocrine Axis. Neuroendocrinology, 2015. 101(3): p. 193-210). Previous reports on the functions of KISS1 R found a critical role of KISS1 R activation in regulating the migratory and metastatic capabilities of various cancers (Guzman ef a / .). Furthermore, studies have found that KISS1R activation was responsible for inducing pubertal development by regulating various hormones’ production and secretion (Millar ef a / .). Since these initial discoveries, the majority of research has investigated KISS1R signaling mechanisms and outcomes largely within neural tissues, sex tissues, or cancers. Within both animal and human populations, several mutations have been identified within both the KISS1 andKISS1R genes that result in a change in binding or signaling efficiency (Teles, M.G., et al., Human diseases associated with GPR54 mutations, Progress in molecular biology and translational science, 2009. 88: p. 33-56; Teles, M.G., et al., A GPR54-activating mutation in a patient with central precocious puberty, New England Journal of Medicine, 2008. 358(7): p. 709-715; Semple, R., et al., Two novel missense mutations in g protein-coupled receptor 54 in a patient with hypogonadotropic hypogonadism, The Journal of Clinical Endocrinology & Metabolism, 2005. 90(3): p. 1849-1855; Silveira, L.G., et al., Mutations of the KISS1 gene in disorders of puberty, The Journal of Clinical Endocrinology & Metabolism, 2010. 95(5): p. 2276-2280). The currently known conditions associated with mutations in either of these genes are precocious puberty and hypogonadotropic hypogonadism (Ke, R., X. Ma, and L.T. Lee, Understanding the functions ofkisspeptin and kisspeptin receptor (Kissi R) from clinical case studies. Peptides, 2019. 120: p. 170019).

[0006] The KISS1 gene codes for a preproprotein that is cleaved as it is trafficked out of the cell (Guzman et al.). When the KISS1 preproprotein is cleaved, it produces one of four common peptides called Kisspeptin Kp-10, Kp-13, Kp-14, and Kp-54 (Guzman et al.). The numerical portion of the name denotes how many amino acid residues remain in the peptide after cleavage, with Kp-10 being the smallest functional peptide. Each of these peptides shares a ten amino acid sequence on the C terminal that has been identified as necessary for interacting with KISS1R (Niida, A., etal., Design and synthesis of downsized metastin (45-54) analogs with maintenance of high GPR54 agonistic activity. Bioorganic & medicinal chemistry letters, 2006 16(1 ): p. 134-137).

[0007] A definite secondary structure has proven to be difficult to construct for the kisspeptins due to the presence of disordered domains (Ibanez de Opakua, A., et al., The metastasis suppressor KISS1 is an intrinsically disordered protein slightly more extended than a random coil, PLoS One, 2017. 12(2): p. e0172507; Gutierrez-Pascual, E., et al., In vivo and in vitro structure-activity relationships and structural conformation of Kisspeptin-10-related peptides, Molecular Pharmacology, 2009. 76(1 ): p. 58- 67). Without the use of in silico modeling, most work involving kisspeptins uses amino acid substitution to identify interactions. Through these studies, it has been found that amino acids Phe1, Arg2, and Phe5are required for Kisspeptin to bind to KISS1 R and that any substituted amino acids must have highly similar properties in order to prevent complete loss of signaling (Niida, A., et al.; Gutierrez-Pascual, E., et al.; Curtis, A.E., et al., A kisspeptin-10 analog with greater in vivo bioactivity than kisspeptin-10, Endocrinology and Metabolism, 2009: p. E296-E303; Roseweir, A.K., et al., Discovery of potent kisspeptin antagonists delineate physiological mechanisms of gonadotropin regulation. Journal of Neuroscience, 2009. 29(12): p. 3920-3929). Similarly, several residues that can tolerate certain substitutions have been identified, such as residues 6 and 10. This tolerance in amino acid properties allows for new peptides to be synthesized for clinical application related to reproductive disorders (Hu, K.-L., et al., Advances in clinical applications of kisspeptin-GnRH pathway in female reproduction. Reproductive Biology and Endocrinology, 2022. 20(1 ): p 81 ).

[0008] The above-cited references provided for background purposes are herein incorporated by reference in their entirety.

[0009] Thus, there is a need in the art for improved therapeutic compositions and methods for the treatment of lung diseases, such as the airway remodeling aspect of asthma, including suchcompositions and methods that would display an anti-proliferative effect on lung tissue while remaining non-cytotoxic.Brief Summary

[0010] Discussed herein are various embodiments of improved therapeutic compositions and methods for treating lung diseases, particularly the remodeling aspect of asthma, with potent anti-proliferative effect on lung tissue, and low cytotoxicity to lung cells and tissues. The novel KPSAT peptides disclosed herein have potent KISS1 R agonist capabilities that potentially increase the beneficial outcomes of KISS1R signaling in the context of treating lung disorders, including asthma.

[0011] Example 1 comprises peptides containing different combinations of at least two amino acid substitutions.

[0012] Example 2 relates to the peptides according to Example 1 , wherein one or more peptides of claim 1 , wherein, relative from an amidated C-terminus, the two or more amino acid substitutions are at positions 6, 10, or 12.

[0013] Example 3 relates to the peptides of Example 1 wherein the peptides have an amino acid sequence selected from the group comprising:SEQ ID NO: 1 : LTNWNWNSFGLRF - NH2;SEQ ID NO: 2: LTNYNWNAFGLRF - NH2; andSEQ ID NO: 3: LPNWNWNAFGLRF - NHz

[0014] Example 4 relates to the peptides of Example 3 wherein at least two sequences of the group are present.

[0015] Example 5 relates to the peptides of Example 3 wherein at least three sequences of the group are present.

[0016] Example 6 relates to a method of treating lung diseases comprising the steps of administering to a subject a therapeutically effective amount of one or more modified Kp-10 or Kp13 peptides having two or more amino acid substitutions.

[0017] Example 7 relates to the method of Example 6 wherein the two or more amino acid substitutions are, relative from an amidated C-terminus, at positions 6, 10 or 12.

[0018] Example 8 relates to the method of Example 6 wherein the one or more modified kp-10 or Kp13 peptides comprise sequences selected from the group consisting of SEQ ID NO: 1-3.

[0019] Example 9 relates to the method of Example 8 wherein at least two sequences of the group are present.

[0020] Example 10 relates to the method of Example 8 wherein at least three members of the group are present.

[0021] Example 11 relates to the method of Example 6, wherein the lung disease is asthma, COED, Lymphangioleiomyomatosis (LAM), bronchiolitis obliterans / popcorn lung, Idiopathic Pulmonary Fibrosis (IPF), pulmonary symptoms of sarcoidosis or other lung remodeling-related diseases.

[0022] Example 12 relates to the method of Example 6 wherein one or more peptides are used in conjunction with bronchodilators, glucocorticoids, B2 adrenergic receptor agonists, muscarinic receptorantagonists, antibody / immunotherapy, leukotriene synthesis inhibitors or leukotriene receptor antagonists.

[0023] Example 13 relates to the method of Example 6 wherein the one or more peptides are administered through intranasal administration.

[0024] Example 14 relates to a method of treating a disease or condition through an increase in KISS1 / KISS1 R agonist function, comprising the steps of administering to a subject a therapeutically effective amount of one or more modified Kp-10 or Kp13 peptides having two or more amino acid substitutions.

[0025] Example 15 relates to the method of Example 14 wherein the disease or condition is one or more of decreased fertility, decreased libido, delayed puberty, obesity or Cytokine Release Syndrome (CRS).

[0026] Example 16 relates to the method of Example 15 wherein the one or more modified Kp-10 or Kp13 peptides are administered intranasally or subcutaneously.

[0027] While multiple embodiments are disclosed, still other embodiments will become apparent to those skilled in the art from the following detailed description, which shows and describes various illustrative implementations. As will be realized, the various embodiments herein are capable of modifications in various obvious aspects, all without departing from the spirit and scope thereof. Accordingly, the drawings and detailed descriptions are to be regarded as illustrative in nature and not restrictiveBrief Description of the Drawings

[0028] FIG. 1 is a peptide sequence alignment of KPSATs with the following peptide sequences: A) Natural Kp-10 sequence derived from the KISS1 gene, B) Natural Kp-13 sequence derived from the KISS1 gene, C) Novel KPSAT1, with Y10W and P12T substitutions, D) Novel KPSAT2 with S6A and P12T substitutions, and E) Novel KPSAT3 with S6A and Y10W substitutions. All sequences aligned with an amidated C-Terminus. Missing amino acid residues are denoted with a Substitutions in KPSATs are highlighted and bolded, according to one embodiment.

[0029] FIGs. 2A-D are bar diagrams of the cytotoxic effects of KPSAT treatments with FIG. 2A showing the effects with Natural Kp-10, FIG. 2B with KPSAT1 , FIG. 2C with KPSAT2, and FIG. 2D with KPSAT3. In each case, Triton-X was used as a positive control to induce total cell death, and fresh serum-free media was used as a negative control All kisspeptins were tested on a log scale to evaluate the effects of 24-hour exposure on cell membrane stress. Cytotoxicity was compared to the negative control, and significance was calculated using one-way ANOVA followed by Tukey’s post-hoc test. Data is shown as Mean ± SEM n=5-6, according to one embodiment.

[0030] FIGs. 3A-C are bar diagrams of the antiproliferative effects of KPSAT treatments, where the effects of Kp-10 are compared to KPSAT1 (FIG. 3A), KPSAT2 (FIG. 3B), or KPSAT3 (FIG. 3C) on basal and PDGF-induced proliferation in ASM cells, and each was evaluated via MTT Assay. Significance was calculated using an unpaired T-test against appropriate Kp-10 controls Data is shown as mean + SEM. n=4-12. *p<0.05, **p<0.01, ***p<0.001.

[0031] FIG. 4 is a bar diagram with superimposed in-well western blot images depicting the effect of KPSAT treatment on the deposition of ECM proteins, such as A) Collagen I, B) Collagen III, and C) Fibronectin. The effect of K p-10 and KPSATs on ASM ECM protein deposition, both in typical conditions and in response to transforming growth factors (TGF-P1), was evaluated using in-well western blot analysis. Significance was calculated using an unpaired T-test against appropriate Kp-10 and TGF-pi controls. Data were analysed using one-way analysis followed by Tukey’s post hoc test. Data presented as mean ± SEM (n=5 / group). ***p<0.001 vs Vehicle,###p<0.001 vs TGF-p1 .

[0032] FIG. 5 illustrates the effect of KPSATs on GMP / MA-induced lung respiratory function in mice. Mice were sensitized with 0.5 g c-di-GMP and mixed allergen (MA: 10 pg each of Ovalbumin, Alternaria alternata, Aspergillus fumigatus, and Dermatophagoides farinae (house dust mite)) or (Dulbecco’s phosphate-buffered saline, DPBS, vehicle) intranasally (i n.) on alternate days (starting from day 1 ) for 4 weeks (3 days / week). From day 2 onward, on alternate days (3 days / week), mice in the respective groups received DPBS or KPSATs by intranasal administration (A) is a schematic showing the experimental design of the study; (B) is the total respiratory system resistance (Rrs); and (C) is the total respiratory system elastance (Ers). Data were analyzed using two-way ANOVA followed by Tukey’s post hoc test. Mean ± SEM (n = 5 mice / group); ** / ##p < 0 01 ; *** / ###p < 0.001 vs Veh or GMP / MA-challenged group.

[0033] FIG. 6 illustrates the effect of KPSATs on GMP / MA-induced airway inflammation. (A) is a representative image of hematoxylin and eosin (H&E)-stained lung sections of WT mice (O57BL / 6) treated with PBS or GMP / MA or KPSATs Images were captured at 20X magnification, a scale bar 60 pm is depicted. Inflammatory cell recruitments in the respective airways is shown in (B); the total number of bronchoalveolar lavage (BAL) cells is shown in (C); and (D) shows the number of neutrophils (D) in the BAL fluid of WT mice challenged with PBS or GMP / MA or KPSATs. The results were scored blindly by independent individuals and plotted using GraphPad prism software. Data were analyzed using oneway ANOVA followed by T ukey’s post hoc test. Mean ± SEM (n = 5 mice / group); #p < 0.05, “p < 0.01 , *** / ###p < Q 001 versus Veh or GMP / MA-challenged group.

[0034] FIG. 7 illustrates the effect of KPSATs on GMP / MA-induced airway collagen deposition. Representative images of Sirius Red / Fast Green stained lung section from WT mice treated with PBS or GMP / MA or GMP / MA with KPSATs, scale bar 60 pm (A). The semiquantitative measurement of the airway collagen deposition was scored by four independent individuals and plotted graph using GraphPad prism software (B). Black arrows indicate areas of high collagen deposition around or near the airways. Data were analyzed using one-way ANOVA followed by Tukey’s post hoc test. Mean ± SEM (n = 5 mice / group); **p < 0.01, *** / ###p < 0.001 versus Veh or GMP / MA-challenged group.Detailed Description

[0035] The various compositions and methods of treatment with those composition embodiments disclosed or contemplated herein include novel KPSAT peptides having a more potent agonist function to KISS1 / KISS1R signaling that increases the beneficial outcomes of Kisspeptin signaling towards novel treatment of lung disease, particularly asthma. In certain embodiments, each of the novelsynthesized peptides have two amino acid substitutions and can treat lung disease by improving therapeutic benefits in regulating pathophysiological changes in lung tissue

[0036] More specifically, the various embodiments herein relate to novel kisspeptins, termed KPSATs, having effects on airway smooth muscle (ASM) cells. These KPSATs show no indicators of cytotoxicity, but show variable changes within antiproliferative capabilities, and comparable effects on regulating ECM deposition compared to unmodified Kp-10. These KPSATs having novel amino acid substitutions alter KISS'! R signaling efficacy. It has been shown that airway smooth muscle (ASM) cells express both KISS1 and KISS1 R at detectable levels. It further shows that KISS1 / KISS1 R signaling has many beneficial outcomes in regulating ASM cell functionality in the context of diseases of the lung, particularly asthma.

[0037] The newly designed peptide embodiments herein have strong affinity to KISS'! R and thus will provide a positive effect in regulating ASM functionality, and thereby disease pathophysiology. Hence, the various embodiments herein relate to increased binding efficiency between KISS'! R and its ligand, thereby improving airway outcomes. More specifically, the three novel Kisspeptin-13’s (referred to as KP-Satellites, KP-SATs or KPSATs) are based on the conserved kisspeptin sequence and have two amino acid substitutions each. The effects of these KP-SATs on ASM are discussed in the Examples below.

[0038] The novel peptides of the various embodiments herein do not display cytotoxic effects and show similar results to Kp-10 Further, the anti-proliferative natures of these peptides have been tested using MTT proliferation assays. Among the three peptides, KPSAT 1 and KPSAT2 exhibited higher antiproliferative effects compared to Kp-10, and KPSAT3 showed anti-proliferative capabilities similar to Kp-10. Furthermore, KPSAT's demonstrated significant effect in regulating the ECM protein production in ASM cells, comparable to that of Kp-10. In addition, these peptides inhibit GMP / MA-induced airway remodeling and airway hyperresponsiveness in a mouse model of asthma. The developed peptides are bioactive and noncytotoxic. The increased anti-proliferative ability exhibited by these peptides highlights that the modifications to the peptides provide an improvement over natural Kp-10 / KISS1Rsignaling.

[0039] One exemplary embodiment provides treatments with these novel kisspeptins to prevent pathologies associated with asthma, COPD, Lymphangioleiomyomatosis (LAM), bronchiolitis obliterans / popcorn lung, Idiopathic Pulmonary Fibrosis (IPF), pulmonary symptoms of sarcoidosis and other lung remodeling-related diseases. In accordance with additional embodiments, this type of therapeutic can be used in conjunction with additional medications to provide relief and slow disease progression for chronic lung diseases such as asthma, COPD, LAM, and pulmonary fibrosis. In additional embodiments, KPSATs are used in conjunction with one or more of bronchodilators, glucocorticoids, B2 adrenergic receptor agonists, muscarinic receptor antagonists, antibody / immunotherapy, leukotriene synthesis inhibitors or leukotriene receptor antagonists.

[0040] In another embodiment, KPSATS are used for reducing inflammation for a subject experiencing Cytokine Release Syndrome (CRS) that resulted from other health conditions or therapeutic interventions.

[0041] In an additional embodiment, a composition for a pulmonary therapeutic is comprised of one or more of KPSAT1 , KPSAT2, and KPSAT3. In an additional embodiment, the modified peptides are administered to a patient through intranasal administration

[0042] In another embodiment, KPSATs are administered to treat decreased fertility, decreased libido, delayed puberty, and for increased weight loss. The KPSAT administration could be through intranasal administration or subcutaneous injection.

[0043] EXAMPLE 1 Methods:

[0044] Peptides: Peptides were synthesized by GenScript (New Jersey, US). Peptides were produced with purity >97%. Peptides were tested to confirm sample sequence, purity, and solubility The following peptides were synthesized:SEQ ID NO: 1 : LTNWNWNSFGLRF - NH2(KPSAT1 );SEQ ID NO: 2: LTNYNWNAFGLRF - NH2(KPSAT2);SEQ ID NO: 3: LPNWNWNAFGLRF - NH2(KPSAT3);SEQ ID NO: 4: — YNWNSFGLRF - NH2; (Kp-10) andSEQ ID NO: 5: LPNYNWNSFGLRF - NH2(Kp-13)

[0045] Cell Culture:

[0046] Human bronchial samples were obtained from patients undergoing thoracic surgery at St. Mary’s Hospital, Mayo Clinic, Rochester (approved by Mayo Clinic IRB, utilized focal non-infectious causes). Airway samples were denuded of epithelium and ASM tissue was enzymatically dissociated to isolate ASM cells. Cells were maintained under standard conditions of 37°C (5% CO2, 95% air) using DMEM / F-12 supplemented with 10% FBS and 1% AbAm. For experiments, ASM cells were limited to the 5th passage in order to preserve the ASM phenotype; Cultures were periodically tested to confirm the contractile phenotype. Cells were serum deprived prior to experiments using serum-free DMEM / F- 12 supplemented with 1% AbAm.

[0047] LDH Cytotoxicity Assay:

[0048] The LDH cytotoxicity assay was performed on two patient-derived ASM lines, with each condition done in triplicate. The LDH kit by BioVision was used (Catalogue #: K311-400). ASM cells were grown to confluency, trypsinized and seeded at 2,000 cells / well. Cells were serum-deprived for 24 hours prior to experimentations. All treatments were delivered in SFM. 1% Triton X-100 was used as a positive control for cell stress and death. Novel KPSATs and Kp-10 were tested at 0.1-10 pM concentrations. Treatments were tested at 24 hours to test for cytotoxicity. All results were made relative to the 24-hour control’s absorbance.

[0049] Proliferation Assays:

[0050] Cells were grown to confluency and trypsinized. Cells were seeded at 7,000 cells / well and serum-deprived 48 hours prior to experimentation. All treatments were delivered in 1% Serum Media, with SFM acting as a control. The effects of Kp-10 and novel KP-SAT’s on proliferation was tested at 0 1 , 1 0 and 5 0 pM concentrations PDGF was used as a mitogen at 4 ng / mL Proliferation was measured using a standard MTT Assay. Absorbances were read at 570 nm using a Synergy HTX MultiMode Plate Reader. All values were made relative to the proliferation observed in the 1% controls.

[0051] ECM Deposition Assay:

[0052] Human ASM cells were allowed to adhere overnight in 96-well black-walled clear-bottom plates and then serum starved for 48 h. Cells were treated in fresh serum-free media with Kp-10 (1 M), Kp- SATs (1pM) and TGF-p1 (2ng / ml) for 48h. Following the treatment period, a total number of cells were counted in each well by high-contrast bright-field direct cell counting by a Lionheart FX Automated Microscope (LFX; BioTek Instruments, Winooski, VT, USA). After cell count, wells were decellularized using 0.016 N NH4OH for 45 min, and cellular detachment was confirmed visually by a microscope, ensuring that only ECM remained. The plate was then washed 3 times with PBS, and nonspecific protein binding was blocked by using 5.0% bovine serum albumin for 1 hour priorto overnight incubation with primary antibodies of collagen I, collagen III, and fibronectin at 4°C. Wells were then washed again and incubated for 60 min with infrared dye-conjugated secondary antibodies. ECM deposition was then measured using a semiquantitative immunofluorescence In-Cell Western technique using Li-Cor Odyssey XL System with densitometry quantification. ECM protein expression was normalized to cell number measured via high-contrast bright-field direct cell counting performed on each plate before cell removal

[0053] GMP / Mixed Allergan (MA)-induced Severe Asthma Mouse Models:

[0054] GMP / MA mice were exposed to a cocktail of allergens containing 10 pg each of ovalbumin, Alternaria alternata, Aspergillus fumigatus and Dermatophagoides farina and 0.5 pg of cyclic-di-GMP in 25 uL of DPBS. MA mice were exposed to the same cocktail, without the cyclic-di-GMP component. Mice were exposed via intranasal administration on alternate days for four weeks (3 days / week). From day 2, mice from all three study populations received ~30 pg / mice of KPSATs (KPSAT1, KPSAT2 & KPSAT3) in 25 pl of DPBS intranasally on alternate days (3 days / week) Administration of KPSATs and MA was performed under isoflurane anesthesia. An overview of the experimental plan is depicted in the schematic in FIG. 5.

[0055] Lung Function Analysis:

[0056] Mice were assessed using a flexiVent system (Scireq, Montreal, Canada) on day 29, and parameters such as total respiratory system resistance (Rrs), and elastance (Ers) were recorded. Ketamine (100 mg / kg i.p.) and xylazine (10 mg / kg i.p.) were used for anesthesia. Rrs, and Ers were recorded at baseline (0 mg / ml Methacholine, MCh) and increasing doses of nebulized MCh (6.25-50 0 mg / ml). Throughout the flexiVent analysis, mice were maintained at 37°C using a heated light source placed above at 45°, with continuous observation of the heart rate.

[0057] Bronchoalveolar Lavage Fluid (BALF) and Lung Tissue Collection:

[0058] After the lung function measurements, 1.0 mL of DPBS (supplemented with a PPI cocktail) was injected into the lungs via tracheal cannula to obtain the BALF. With a 60% recovery rate, approximately 600 pL of fluid was collected in the tubes. BALF underwent processing to separate immune cells and supernatant The collected BALF supernatant was stored at -80°C until analysis Isolated immune cells were fixed and stained for differential counts Following this, the lungs were perfused with 10 mL of DPBS via the right ventricle to eliminate circulating blood before collecting each lobe The left lobe of each mouse lung was stored in Carnoy’s solution (100% ethanol, chloroform, and glacial acetic acid in a ratio of 6:3:1 with ferric chloride, (Sigma-Aldrich) for a few hours, followed by the replacement of the solution with 70% ethanol and preparation for histology studies.

[0059] Total and Differential Leukocyte Count in BALF:

[0060] The collected BALF from the respective groups of mice was processed for total and differential leukocyte count (DLC). BALF samples were centrifuged at 2,000 rpm for 5 min at 4 °C. The supernatant was retained for cytokine analysis and the cell pellet was mixed with 100 pl of ACK Lysing Buffer (Cat# A1049201, Thermofisher Scientific) to lyse red blood cells. Following another centrifugation, the cell pellet was resuspended in 200 L of PBS, followed by performing a total BAL cell count using Countess- II FL cell counter (Thermo Fisher Scientific). The remaining sample was processed using a cytospin on glass slides and the resultant air-dried smear was stained using the Differential Quick Staining kit (Cat# 26096-50, Modified Giemsa, EMS) following the manufacturer’s protocol. The processed slides were then observed by an inverted phase contrast microscope (Accu-Scope EXI-410) at X40 magnification for DLC. The actual cell count number for the total BAL cell count is provided. A minimum of 100 cells per sample were counted for differential leukocyte counts and then converted to an absolute value based on the total number of cells in the respective samples.

[0061] Histopathology Analyses:The stored left lung lobes were embedded in paraffin wax blocks and sections were stained using H&E (Sigma-Aldrich), PAS (EMS, Hatfield, PA, USA) and Sirius Red / Fast Green (Chondrex, Redmond, WA, USA), using previously described techniques. The slides were then scanned using Epredia Pannoramic MIDI II 20x (3DHISTECH, Budapest, Hungary). The scanned H&E sections were examined for inflammatory cell recruitment around the airways and Collagen was stained with Sirius Red / Fast Green to examine the positively-stained area around the airways. The inflammatory cell recruitment and collagen deposition was determined by blind scores by four independent individuals.EXAMPLE 2

[0062] KPSAT Sequences: Peptides containing different combinations of at least two amino acid substitutions were generated. Aligned sequences showing the KPSAT peptides with these specific substitutions KPSAT1 , KPSAT2, and KPSAT3 can be found in FIG. 1 along with reference sequences Kp-10 and Kp-13. Throughout, any references to an amino acid location is relative from the amidated C-terminus (‘T being phenylalanine, ‘F’).EXAMPLE 3

[0063] KPSAT peptide cytotoxicity effect as shown by LDH Cytotoxicity Assay: Lactate Dehydrogenase (LDH) is a ubiquitous enzyme between human cell types. LDH exists within the cytosol of the cell but is leaked into the extracellular environment during periods of stress on the membrane and during cell death. Relative supernatant concentrations of LDH can be quantified to determine if a compound or treatment induces stress on a cell. ASM cells were seeded with a uniform density within this assay to make culture supernatants comparable. Triton-X was added as a positive control due to its capabilities in solubilizing cell membranes and inducing cell death. Serum-free media was used as a negative control to compare typical LDH release to specific treatments. After adjusting for background and making LDH concentrations relative between the negative and positive control, the effects of specific Kisspeptins was analyzed.

[0064] It has been demonstrated previously (data not shown) that Kp-10 showed little elevation in supernatant LDH content compared to the negative control. The assays performed here validated theseprevious findings, showing treatment with Kp-10 did not increase LDH release in any significant quantity (FIG. 2A). Three concentrations using a log dose scale were tested, focusing on the concentrations found within most kisspeptin research. Each of the KPSATs were also evaluated for effects on LDH release to ensure that the substitutions did not inadvertently make the synthetic peptide cytotoxic. In comparison to the negative control, each of the KPSATs did not significantly affect LDH release (FIGs. 2B-D).EXAMPLE 4

[0065] Proliferation Assay: Rates of cell growth can be influenced by altering different internal signaling pathways. Kp-10 is known to have an inhibitory effect on proliferation, with this being a well- defined interaction observed in many cell types. Cells are typically serum deprived prior to a proliferation assay to synchronize the cell’s growth phases and increase homogeneity within their response. Low concentrations of serum (1%) are added into media in a proliferation assay in order initiate basal proliferation rates. Additionally, the use of a mitogen, or proliferation-stimulating compound, is typical in order to compare the effects of a treatment in different circumstances. In this experiment, Platelet- Derived Growth Factor (PDGF) was used as the mitogen.

[0066] Within these assay results, different combinations of kisspeptins and PDGF were tested for the proliferation effect of kisspeptins These results for Kp-10 were similar to initial studies. Kp-10 mildly blunted basal proliferation and significantly blunted PDGF-induced proliferation. The effect of each of the KPSATswas compared to appropriate Kp-10 controls (FIG. 3A-C). KPSAT1 significantly decreased the proliferation rates of both basal and PDGF-induced proliferations beyond KP-10’s effects (FIG. 3A). KPSAT2 had an increased effect in reducing PDGF-induced proliferation comparison to Kp-10 (FIG. 3B). KPSAT3 had antiproliferative effects but was not significantly different from Kp-10 (FIG. 3C).EXAMPLE 5

[0067] ECM Deposition: Airway smooth muscle cells secrete different mixes of collagens and structural proteins referred to as the Extracellular Matrix (ECM). Airway smooth muscle cells typically synthesize additional ECM components while simultaneously breaking down older components, with this process being referred to as ECM turnover. Multiple signaling mechanisms are involved within ECM regulation. As mentioned above, during asthma or in the presence of continuous or prolonged inflammation, there is more ECM deposition which increases the stiffness of the airway as different collagens and structural proteins increase in prevalence. To determine the potential therapeutic effect of kisspeptins / KISSIR signaling on ECM regulation, an in-well western assay was performed. In this assay, ECM deposition isis quantified under normal conditions and compared to ECM production in response to a growth factor like TGF-p1

[0068] Results show that Kp-10 significantly blunted the production of collagen I, collagen III, and fibronectin in ASM cells treated with TGF-p1 (FIG. 4). Treatments with each of the KPSATs did not show any significant changes in the production of the three ECM components compared to Kp-10 or vehicle in basal conditions However, each of the KPSATs were effective in reducing the production of the ECM components during TGF-p1 treatments.

[0069] Effect of KPSATs on total respiratory system resistance (Rrs): The lung functions were assessed using the forced oscillation technique by the flexiVent FX1 module with an in-line nebulizer(SCIREQ, Montreal, Canada). WT mice showed a dose-dependent linear increase in Rrs (representing airway constriction) in response to nebulized methacholine concentrations (MCh; 0-50 mg / mL) in both PBS and GMP / MA-challenged groups (FIG. 5B). For comparison between the different groups, Rrs at the concentration of 50 mg / mL of MCh was considered GMP / MA-challenged mice showed a significant increase in max Rrs (p<0.001 ; FIG. 5B) compared to their respective PBS-challenged group. In comparison between GMP / MA-challenged mice and KPSATs treatment groups at a concentration of 50 mg / mL of MCh, although KPSAT1 and KPSAT2 showed significant protective effects against GMP / MA treatment, the protective effect of KPSAT3 is higher among the KPSATs treatment groups in GMP / MA-challenged mice.

[0070] Effect of KPSATs on total respiratory system elastance (Ers): Ers was measured for the lungs of GMP / MA-challenged mice and the effect of KPSATs was assessed in the respective treatment groups. We observed an increase in Ers in GMP / MA-challenged mice compared with DPBS (FIG. 5C). Max Ers in response to 50 mg / ml MCh was used to compare the effectiveness of the treatments. GMP / MA-challenged mice showed a significant increase in max Ers (p<0.001 ; Figure 5C) compared to their respective PBS-challenged group. In comparison between GMP / MA-challenged mice and KPSATs treatment groups of MCh, although KPSAT1 and KPSAT2 showed a significant decrease in Ers at a concentration of 50 mg / mL, KPSAT3 elicited a significant decrease in Ers among the KPSATs treatment groups in GMP / MA-challenged mice.

[0071] Effect of Kissi R on airway structure and morphology: To assess KPSATs protective role on airway inflammation and structure in GMP / MA-challenged mice, staining was performed on the lung sections of mice challenged with PBS or GMP / MA or KPSATs treatment with H&E (FIG. 6A) stain. GMP / MA-challenge significantly induced airway inflammation with immune cell infiltration (p<0.001 ; FIG. 6B) compared to respective PBS-challenged groups. In comparison between KPSATs treatment mice, we observed a significant decrease in airway immune cell infiltration with all KPSATs treatments, however KPSAT 1 showed significant (p<0.001 ) decrease in inflammatory cell recruitment compared to KPSAT2 (p<0 05; FIG. 6B) and KPSAT3 (p < 0.01 ; FIG. 6B). We further measured the total BAL cell count and found a significant increase in total BALF cell count after the GMP / MA-challenged mice (p<0.001 ; FIG. 6C). The protective effect of KPSAT3 in decreasing immune cell infiltration was more profound (p<0.001; Figure 6C) compared to KPSAT1 (p<0.01; FIG. 6C) and KPSAT2 (p<0.05; FIG. 6C).

[0072] In addition, it was determined whether these KPSATs had any effect on specific immune cell types by differentially counting the immune cells in BAL samples. Consistent with the above finding, the GMP / MA challenge significantly increased the number of neutrophils in BAL samples (p < 0.001; FIG. 6D) compared to respective PBS-challenged groups. In a comparison between the treatment groups, GMP / MA-challenged and treated with KPSAT3 showed a significant decrease in neutrophil (p<0.001 ; FIG. 6D) count compared to GMP / MA-challenged and KPSAT1 (p<0.01 ; FIG. 6D) and KPSAT2 (p<0 01 ; FIG 6D) treated mice

[0073] Effect of KPSATs on regulating airway collagen deposition: Sirius Red / Fast Green staining was done in the lung section of GMP / MA-challenged mice or mice challenged with PBS or treated with KPSATs for a qualitative evaluation of collagen deposition in the airways (FIG. 7A). GMP / MA-challenged mice groups showed a significant (p<0.001 ; FIG. 7B) increase in collagen deposition around the airways compared to their respective PBS-challenged group. Notably, the effect of KPSAT3 on GMP / MA-challenged on collagen deposition around the airways was observed significantly (p<0.001; FIG 7B) higher compared to KPSAT1 or KPSAT2 group. Also, KPSAT1 (p<0.01 ; FIG. 7B) and KPSAT2 (p<0.01 ; FIG. 7B) showed a significant effect on reducing collagen deposition on the airways induced by GMP / MA treatment

[0074] The present inventive subject matter provides among other embodiments novel KP-13s peptides with dual amino acid substitutions. These novel peptides do not display cytotoxic effects and showed similar results to Kp-10. Further, the anti-proliferative nature of these peptides were tested using MTT proliferation assays. Among the three embodiments of peptides, KPSAT1 and KPSAT2 exhibited higher anti-proliferative effect compared to Kp-10, whereas KPSAT3 showed antiproliferative capabilities similar to Kp-10. To test KPSATs anti-fibrotic effect, in -well western assay were performed. Each of the KPSATs showed similar anti-fibrotic effect compared to Kp-10. Altogether, these results suggest that the developed peptides are bioactive and non-cytotoxic.

[0075] While the various systems described above are separate implementations, any of the individual components, mechanisms, or devices, and related features and functionality, within the various system embodiments described in detail above can be incorporated into any of the other system embodiments herein.

[0076] The terms “about” and “substantially,” as used herein, refers to variation that can occur (including in numerical quantity or structure), for example, through typical measuring techniques and equipment, with respect to any quantifiable variable, including, but not limited to, mass, volume, time, distance, wave length, frequency, voltage, current, and electromagnetic field. Further, there is certain inadvertent error and variation in the real world that is likely through differences in the manufacture, source, or precision of the components used to make the various components or carry out the methods and the like. The terms “about” and “substantially” also encompass these variations The term “about” and “substantially” can include any variation of 5% or 10%, or any amount - including any integer - between 0% and 10%. Further, whether or not modified by the term “about” or “substantially,” the claims include equivalents to the quantities or amounts.

[0077] Numeric ranges recited within the specification are inclusive of the numbers defining the range and include each integer within the defined range. Throughout this disclosure, various aspects of this disclosure are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges, fractions, and individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc , as well as individual numbers within that range, for example, 1 , 2, 3, 4, 5, and 6, and decimals and fractions, for example, 1.2, 3.8, 1 ! , and 4% This applies regardless of the breadth of the range

[0078] Although the various embodiments have been described with reference to preferred implementations, persons skilled in the art will recognize that changes may be made in form and detail without departing from the spirit and scope thereof.

Claims

ClaimsWhat is claimed is:

1. One or more peptides comprising modified Kp-10 or Kp13 peptide sequences having two or more amino acid substitutions and having KISS1 / KISS1R agonist function.2 The one or more peptides of claim 1 wherein, relative from an amidated C-terminus, the two or more amino acid substitutions are at positions 6, 10 or 12.

3. The one or more peptides of claim 1 wherein the one or more peptides have an amino acid sequence selected from the group comprising:SEQ ID NO: 1 : LTNWNWNSFGLRF - NH2;SEQ ID NO: 2: LTNYNWNAFGLRF - NH2; andSEQ ID NO: 3: LPNWNWNAFGLRF - NH4. The one or more peptides of claim 3 wherein at least two sequences of the group are present.

5. The one or more peptides of claim 3 wherein at least three sequences of the group are present.

6. A method of treating lung diseases comprising the steps of : administering to a subject a therapeutically effective amount of one or more modified Kp-10 or Kp13 peptides having two or more amino acid substitutions.

7. The method of claim 6 wherein the two or more amino acid substitutions are, relative from an amidated C-terminus, at positions 6, 10 or 12.

8. The method of claim 6 wherein the one or more modified kp-10 or Kp13 peptides comprise sequences selected from the group consisting of:SEQ ID NO: 1 : LTNWNWNSFGLRF - NH2;SEQ ID NO: 2: LTNYNWNAFGLRF - NH2; andSEQ ID NO: 3: LPNWNWNAFGLRF - NHz9 The method of claim 8 wherein at least two sequences of the group are present10. The method of claim 8 wherein at least three members of the group are present.

11. The method of claim 6, wherein the lung disease is asthma, COPD, Lymphangioleiomyomatosis (LAM), bronchiolitis obliterans / popcorn lung, Idiopathic Pulmonary Fibrosis (IPF), pulmonary symptoms of sarcoidosis or other lung remodeling-related diseases.

12. The method of claim 6, wherein the one or more peptides are used in conjunction with bronchodilators, glucocorticoids, B2 adrenergic receptor agonists, muscarinic receptor antagonists, antibody / immunotherapy, leukotriene synthesis inhibitors or leukotriene receptor antagonists.

13. The method of claim 6 wherein the one or more peptides are administered through intranasal administration14. The method of claim 6 wherein at least one of the modified Kp-10 or Kp13 peptides are attached to a pharmaceutical carrier.

15. A method of treating a disease or condition through an increase in KISS1 / KISS1R agonist function comprising the steps of: administering to a subject a therapeutically effective amount of one or more modified Kp-10 or Kp13 peptides having two or more amino acid substitutions.

16. The method of claim 15 wherein the disease or condition is one or more of decreased fertility, decreased libido, delayed puberty, obesity or Cytokine Release Syndrome (CRS).

17. The method of claim 16 wherein the one or more modified Kp-10 or Kp13 peptides are administered intranasally or subcutaneously.

18. The method of claim 15 wherein at least one of the modified Kp-10 or Kp13 peptides is modified at, relative from an amidated C-terminus, one or more of positions 6, 10 or 12.

19. The method of claim 15 wherein the one or more modified Kp-10 or Kp13 peptides are selected from SEQ ID NO: 1 , SEQ ID NO: 2 or SEQ ID NO: 3.

20. The method of claim 15 wherein at least one of the modified Kp-10 or Kp13 peptides are attached to a pharmaceutical carrier.

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