Synthetic peptides, prodrugs, pharmaceutical compositions and uses

文档序号：704413 发布日期：2021-04-13 浏览：17次中文

阅读说明：本技术 合成肽、前药、药物组合物及用途 (Synthetic peptides, prodrugs, pharmaceutical compositions and uses ) 是由 C·V·M·F·多纳西门托 D·A·R·S·达席尔瓦 P·V·B·达席尔瓦 M·E·L·P·加于 2019-07-04 设计创作，主要内容包括：本发明涉及平滑肌张力调节合成肽。其还指含有此类肽的药物组合物及其在治疗其中平滑肌张力的调节是有益的紊乱中的用途。(The present invention relates to smooth muscle tone-modulating synthetic peptides. It also refers to pharmaceutical compositions containing such peptides and their use in the treatment of disorders in which modulation of smooth muscle tone is beneficial.)

1. A peptide comprising a pharmacologically active peptide sequence of formula (2):

Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Ile-Aia-Trp-Xaa9-Xaa1O-Xaa11-Xaa12-Xaa13-Xaa14-Xaa15(2)，

amino to carboxyl direction is from left to right;

wherein:

xaa1, Xaa2, and Xaa15 are each independently absent or Ala, Arg, Lys, or His;

xaa3 is independently absent or Ala, Phe, Trp, or Tyr;

xaa4, Xaa5, and Xaa9 are each independently absent or Phe, Trp, or Tyr;

xaa10 is independently absent or is His, Lys, or Arg;

xaa11 is absent or is Ala, Gly, Val, Leu, Iie, Pro, Cys or Met;

xaa12 is absent or Ala; and

xaa13 and Xaa14 are each independently absent or Asn, Gin, Ser, or Thr;

wherein the pharmacologically active peptide sequence of formula (2) has 5 or more consecutive amino acid residues.

2. The peptide according to claim 1, wherein in formula (2), one or more amino acid residues of the group consisting of Xaa1, Xaa2, Xaa3, Xaa4, Xaa5, Xaa11, Xaa12, Xaa13, Xaa14, and Xaa15 are absent.

3. The peptide of claim 2, wherein the pharmacologically active peptide sequence of formula (2) is SEQ ID NO 17, SEQ ID NO 18, SEQ ID NO 19, SEQ ID NO 20, SEQ ID NO 22, SEQ ID NO 23, SEQ ID NO 24 or SEQ ID NO 5.

4. The peptide of claim 1, wherein the pharmacologically active peptide sequence of formula (2) has 15 to 18 consecutive amino acid residues.

5. The peptide according to claim 1, said pharmacologically active peptide sequence of formula (2) having 18 consecutive amino acid residues.

6. The peptide of claim 1, further comprising at least a second peptide or protein.

7. The peptide of claim 1, comprising a peptide of formula (1)

–Z–X–Z’– (1)

Wherein:

x is a pharmacologically active peptide sequence of formula (2) as defined above; and

z and Z' are each peptides independently comprising a cell permeation enhancing amino acid sequence or an activity enhancing amino acid sequence having 2 to 15 naturally occurring amino acids.

8. The peptide of claim 7, wherein H, acetyl, chloride, or trifluoroacetyl is covalently bound to the N-terminus of-Z-X-Z'.

9. The peptide of claim 7, wherein OH or NH2 is covalently bound to the C-terminus of-Z-X-Z'.

10. The peptide according to claim 7, wherein Z is a peptide comprising a sequence containing amino acid residues Gly, Glu, and Arg, respectively, from N-terminus to C-terminus, X has a pharmacologically active peptide sequence of SEQ ID NO. 5, and Z' is absent.

11. The peptide of claim 1, wherein the peptide is linked to a half-life enhancing moiety selected from the group consisting of albumin binding moieties.

12. The peptide of claim 11, wherein the N-terminus is covalently bound to acetyl, and wherein the C-terminus is covalently bound to NH 2.

13. The peptide of claim 1, comprising in multimeric form two or more peptides having the sequence of formula (2) separated by a cleavable linker based on amino acids.

14. A pharmaceutical composition comprising one or more peptides as defined in any one of claims 1 and a pharmaceutically acceptable excipient.

15. A method of making a pharmaceutical composition comprising introducing one or more peptides of claim 1 to a pharmaceutically acceptable excipient, wherein one or more of the peptides are present in an amount sufficient to treat a condition in which modulation of smooth muscle tone is beneficial.

16. The method of claim 15, wherein the disorder is selected from the group consisting of: erectile Dysfunction (ED), Female Sexual Dysfunction (FSD), Benign Prostatic Hyperplasia (BPH), raynaud's syndrome, Pulmonary Arterial Hypertension (PAH), Systemic Arterial Hypertension (SAH) and hyperreactivity of the airways associated with asthma, COPD, pulmonary fibrosis, silicosis, allergic bronchopulmonary aspergillosis, hereditary angioedema, and neonatal hypoxic respiratory failure.

17. The method of claim 16, wherein the peptide has the amino acid sequence of SEQ ID NO 17, SEQ ID NO 18, SEQ ID NO 19, SEQ ID NO 20, SEQ ID NO 22, SEQ ID NO 23, or SEQ ID NO 24, and further wherein the disorder is PAH.

18. A method for treating a disorder in a patient in need of modulation of smooth muscle tone comprising administering to the patient a therapeutically effective amount of the peptide of claim 1.

19. The method of claim 18, wherein the disorder is selected from the group consisting of: erectile Dysfunction (ED), Female Sexual Dysfunction (FSD), Benign Prostatic Hyperplasia (BPH), raynaud's syndrome, Pulmonary Arterial Hypertension (PAH), Systemic Arterial Hypertension (SAH) and hyperreactivity of the airways associated with asthma, COPD, pulmonary fibrosis, silicosis, allergic bronchopulmonary aspergillosis, hereditary angioedema, and neonatal hypoxic respiratory failure.

20. The method of claim 19, wherein the composition has an amino acid sequence of SEQ ID No. 17, SEQ ID No. 18, SEQ ID No. 19, SEQ ID No. 20, SEQ ID No. 22, SEQ ID No. 23, or SEQ ID No. 24, and further wherein the disorder is PAH.

Technical Field

The present invention relates to synthetic peptides and compositions thereof that are modulators of smooth muscle tone. It also relates to pharmaceutical compositions containing such peptides and their use in therapy where modulation of smooth muscle tone is beneficial.

Background

Smooth muscle tissue is an important structural component and regulator of function of various organs and systems. Smooth muscle contractile disorders are associated with a range of clinical manifestations, particularly diseases of the respiratory, vascular, urogenital and gastrointestinal tracts.

Different mechanisms control the tension of smooth muscle in response to local or systemic stimuli. Modulators are produced by adjacent structures, such as Nitric Oxide (NO), to fine tune the tension at a local level. Complementarily, the Autonomic Nervous System (ANS) modulates smooth muscle contractility through the action of sympathetic or parasympathetic fibers in response to stimuli perceived and processed by the Central Nervous System (CNS). However, neurotransmitter receptors are differentially expressed in smooth muscle tissue based on the macrostructure into which such cells are inserted. Thus, pharmacological control of smooth muscle tone differs significantly in diseases of the respiratory, vascular and genitourinary systems. Pharmacological approaches may even produce opposite effects (i.e. contraction and relaxation) in different systems.

Smooth muscle cells are located on the walls of all vessels except capillary and pericellular venules. Smooth muscle, if present, is the primary regulator of vasodilation and thus of blood vessel diameter. Thus, changes in smooth muscle tone determine the vessel diameter, greatly affecting the peripheral resistance and hence blood flow. In pathological conditions, vascular smooth muscle is associated with the development and progression of arterial hypertension, and therefore, they are also targeted by therapeutic approaches that reduce resistance to blood flow. Pharmacological classes (pharmacological classes) used for such purposes include sympathetic ANS stimulation blockers (adrenergic antagonists), angiotensin converting agent inhibitors or NO donor vasodilators.

Similarly, the corpus cavernosum-the vascular structure directly involved in the dilation of the penis-is also made up of layers of smooth muscle tissue. Erection is a neurovascular event that depends on the integrity of the blood vessels, muscles and neuronal structures that make up the penis. When stimulated, nerve endings adjacent to the corpus cavernosum and endothelial cells covering those vessels release NO. Subsequent amplification of the signal cascade results in relaxation of the cavernous smooth muscle, increased blood flow into these structures, and ultimately erection. Failure of this mechanism can lead to impaired erection, insufficient for intercourse, characterized by Erectile Dysfunction (ED).

The goal of reversal therapy of ED is relaxation of the cavernosal smooth muscle and subsequent increase of blood flow within the cavernosal. In this regard, phosphodiesterase-5 inhibitors (PDE5i) include first line of treatment (first line of treatment). However, 36% of patients are clearly resistant or intolerant to PDE5i and must use other pharmacological options. In general, replacement therapy is limited in effectiveness and inconvenient in both administration (i.e., intra-urethral or intracavernosal) and side effects, and is therefore associated with high drug withdrawal rates. However, both gold standard therapy and second line therapy promote increased blood flow into the corpus cavernosum by relaxing the cavernosum smooth muscle. Thus, the medical need for a drug that can produce the same effect in a more convenient manner has not been met.

In the respiratory tract, smooth muscle cells integrate the upper airway and the entire tracheobronchial tree, actively regulating the caliber of these structures, and thus the airflow. Smooth muscle tone in physiological conditions is also regulated by local modulators and by the ANS. However, in contrast to what happens, for example, in the corpus cavernosum, sympathetic signals promote relaxation of the airway and increase the caliber of the airway. In addition, in this case, the pharmacology of the adrenergic and cholinergic pathways underlies the regulation of the contractility of the airways in pathological situations.

Smooth muscle tissue is the central effector of bronchoconstriction associated with hyperreactivity of the airways that is characteristic of inflammatory diseases of the respiratory tract. In these cases, hyperplasia and hypertrophy of the smooth muscle layer is often observed, which contributes to thickening and increased airway contractility. Integration of beta 2-adrenergic agonists is used to rescue the standard treatment of acute bronchoconstriction in patients with asthma or Chronic Obstructive Pulmonary Disease (COPD). This treatment is particularly effective in view of its ability to prevent smooth muscle contraction and induce smooth muscle relaxation in the airways. Following the same principle of reduced contractility, muscarinic antagonists and phosphodiesterase 4 inhibitors (PDE4i) are used as second and third line therapy, particularly for exacerbations associated with COPD.

The peptides described herein are capable of relaxing smooth muscle of different anatomical structures and thus have pleiotropic mechanisms of action. They are therefore useful in the treatment of diseases involving smooth muscle disorders in the blood vessels, gastrointestinal tract, respiratory tract and urogenital tract.

The technical science literature includes reports on peptides having biological activities similar to those described herein. For example, the toxin of the brazilian spider (Phoneutria nigriper) [ south american banana spider ], commonly known as brazilian spider, is rich in biologically active polypeptides with different pharmacological effects. Among the symptoms caused by stinging, priapism observed in male victims is of interest from a pharmacological point of view. Patent application No. PI 0800596 filed in brazil in 2008 describes the role of PnTx2-6 in the erectile function of rats. The patent family derived from CN101585872 also describes the therapeutic potential of PnTx-6 toxin in the treatment of erectile dysfunction.

Subsequently, PnTx2-6 has also been shown to restore erectile function in hypertensive animals (DOCA-sal) and diabetic mice or older rats (for revision, see: NUNES, K.P.CARDOSO, F.L, CARDOSO-Jr., H.C, PIMENTA, A.M.C, De LIMA, M.E.animal toxins as potential pharmacological toxins for a project of interest dynamic function in: Animal toxins of the fact.A.,LANZA,L.F.,CORTES,S.F.,CORDEIRO,M.N.,RICHARDSON,M.,PIMENTA,A.M.,WEBB,R.C.,LEITE,R.,DE LIMA,M.E.Tx2-6 toxin of the Phoneutria nigriventer spider potentiates rat erectile function.Toxicon,51(7):197-206,2008；ANTUNES,A.A.,ISCAIFE,A.,REIS,S.T.,ALBERTINI,A.,NUNES,M.A.,LUCON,A.M.,NAHAS,W.C.,SROUGI,M.Can we predict which patients will experience resolution of detrusor overactivity after transurethral resection of the prostate？The Journal of Urology,9(10):2574-81,2012). These effects appear to be mediated by the activation of the enzyme Nitric Oxide Synthase (NOS) and the release of NO (Yonamine, C.M., TRONCONE, L.R., CAMILLO, M.A. Block of neural nitrile oxide synthase copolymers of Tx2-5, a leaving phosphorus nitride divider toxin.Toxicon,44,169-172,2004；NUNES,K.P.,A.,LANZA,L.F.,CORTES,S.F.,CORDEIRO,M.N.,RICHARDSON,M.,PIMENTA,A.M.,WEBB,R.C.,LEITE,R.,DE LIMA,M.E.Tx2-6 toxin of the Phoneutria nigriventer spider potentiates rat erectile function.Toxicon1197-206, 2008, NUNES, K.P.CARDOSO, F.L, CARDOSO-Jr., H.C, PIMERTA, A.M.C, De LIMA, M.E.animal biology tools for the purpose of manipulating emulsion dynamics. in: Animal oxygen: State of the area.perspectives in Health and Biotechnology.MarElia ena LIMA, Adriano Monteiro De Castro Pimenta, Marie France Martin-Euchai, Russula Benedeta Zingali and Herv. Furthermore, it was proposed that some genes involved in the NO pathway are expressed in the erectile tissue of mice after treatment with the toxin PnTx2-6 (vilranova f.e., ANDRADE e., LEAL e., ANDRADE, p.m., BORRA, r.c., TRONCONE, l.r.,L.,LEITE,K.R.,PARANHOS,M.,CLARO,J.,SROUGI,M.Erection induced by Tx2-6 toxin of Phoneutria nigriventer spider:expression profile of genes in the nitric oxide pathway of penile tissue of mice.Toxicon.54(6),793-801,2009.). The patent US 9,279,004 describes the peptide PnTx (19) constructed from the toxin PnTx2-6, which is a 19 amino acid derivativeBiological, molecular weight 2,485.85 Da. The publication discloses that the peptides are capable of enhancing erectile function as demonstrated by ex vivo induction of relaxation of the mouse cavernous bands. Thereafter, Silva et al (Silva, c.n., nus, k.p., TORRES, f.s., CASSOLI, j.s., SANTOS, d.m., ALMEIDA, fde.m., MATAVEL, a., CRUZ, j.s., SANTOS-MIRANDA, a., NUNES, a.d., casstro, c.h., machandio DER.A.,C.,S.S.,FELICORI,L.,RESENDE,J.M.,CAMARGOS,E.R.,BORGES,M.H.,CORDEIRO,M.N.,PEIGNEUR,S.,TYTGAT,J.,DE LIMA,M.E.PnPP19,a synthetic and nontoxic peptide designed from a Phoneutria nigriventer Toxin,potentiates erectile function via NO/cGMP.J Ural(ii) a 194(5):1481-90.2015) demonstrated that vasodilation promoted by PnTx (19) is mediated by the activation of NOS and the production of NO, in particular by neurons and inducible isoforms of NOS. Thus, PnTx (19) is claimed as a potential candidate for ED therapy with potential for patients refractory to PDE5 i-based therapy.

The smooth muscle tone-modulating peptides of the present invention are more effective than those reported in the prior art. As an example, the peptides were evaluated against PnTx (19) in an experimental model of smooth muscle contractility that has been established in the scientific literature. As will be further shown, the smooth muscle modulator peptides showed significant activity, with the comparator (PnTx (19)) being either completely inactive (smooth muscle of the airways) or having significantly poor activity (smooth muscle of the corpora cavernosa). In addition, PnTx (19) is shown to play a pro-inflammatory role in airway smooth muscle. Inflammation is an important part of the pathophysiology of many diseases, among which lung diseases such as asthma and COPD inflammation as a mechanism to cause or amplify the exacerbations of said diseases. Thus, the use of pro-inflammatory compounds is currently prohibited in such lung diseases. The peptides of the invention do not have a pro-inflammatory effect, but rather inhibit inflammation in the pulmonary system. Furthermore, the action of the peptides described herein is mediated by NO, a pleiotropic mechanism that increases the breadth of potential therapeutic applications.

Disclosure of Invention

In some embodiments, the peptide comprises a pharmacologically active peptide sequence of formula (2): xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Ile-Aia-Trp-Xaa9-Xaa1O-Xaa11-Xaa12-Xaa13-Xaa14-Xaa15(2), wherein the amino to carboxyl direction is from left to right;

wherein:

xaa1, Xaa2, and Xaa15 are each independently absent or Ala, Arg, Lys, or His;

xaa3 is independently absent or Ala, Phe, Trp, or Tyr;

xaa4, Xaa5, and Xaa9 are each independently absent or Phe, Trp, or Tyr;

xaa10 is independently absent or is His, Lys, or Arg;

xaa11 is absent or is Ala, Gly, Val, Leu, Iie, Pro, Cys or Met;

xaa12 is absent or Ala; and

xaa13 and Xaa14 are each independently absent or Asn, Gin, Ser, or Thr;

wherein the pharmacologically active peptide sequence of formula (2) has 5 or more consecutive amino acid residues.

In some embodiments, in formula (2), there is no more than one amino acid residue of the group consisting of Xaa1, Xaa2, Xaa3, Xaa4, Xaa5, Xaa11, Xaa12, Xaa13, Xaa14, and Xaa 15.

In some embodiments, the pharmacologically active peptide sequence of formula (2) is SEQ ID NO 17, SEQ ID NO 18, SEQ ID NO 19, SEQ ID NO 20, SEQ ID NO 22, SEQ ID NO 23, SEQ ID NO 24 or SEQ ID NO 5.

In some embodiments, the pharmacologically active peptide sequence of formula (2) has 15 to 18 consecutive amino acid residues.

In some embodiments, the pharmacologically active peptide sequence of formula (2) has 18 contiguous amino acid residues.

In some embodiments, the peptide comprises at least a second peptide or protein.

In some embodiments, the peptide comprises a peptide of formula (1)

–Z–X–Z’– (1)

Wherein:

x is a pharmacologically active peptide sequence of formula (2) as defined above; and

z and Z' are each peptides independently comprising a cell permeation enhancing amino acid sequence or an activity enhancing amino acid sequence having 2 to 15 naturally occurring amino acids.

In some embodiments, H, acetyl, chloride, or trifluoroacetyl is covalently bound to the N-terminus of-Z-X-Z'.

In some embodiments, OH or NH2 is covalently bound to the C-terminus of-Z-X-Z'.

In some embodiments, Z is a peptide comprising a sequence containing amino acid residues Gly, Glu, and Arg, respectively, from the N-terminus to the C-terminus, a pharmacologically active peptide sequence having SEQ ID NO 5, and Z' is absent.

In some embodiments, any of the peptides described above is linked to a half-life enhancing moiety selected from an albumin binding moiety. In some embodiments, the peptide is a peptide wherein the N-terminus is acetyl and the C-terminus is NH 2.

In some embodiments, the peptide comprises two or more peptides having the sequence of formula (2) separated by an amino acid-based cleavable linker, e.g., by esterification of the C-terminal domain, in multimeric form, wherein the multimer is optionally N-terminally acetylated and C-terminally amidated.

In some embodiments, the invention includes a pharmaceutical composition comprising a smooth muscle tone-modulating peptide and a pharmaceutically acceptable carrier, excipient, or additive for treating a disease associated with aberrant regulation of smooth muscle contractility. For example, in some embodiments, the pharmaceutical composition comprises more than one peptide as defined above and a pharmaceutically acceptable excipient.

In some embodiments, the invention includes the use of smooth muscle tone-modulating peptides for the treatment of diseases benefiting from modulation of smooth muscle contractility, including, but not limited to: erectile Dysfunction (ED), Female Sexual Dysfunction (FSD), Benign Prostatic Hyperplasia (BPH), raynaud's syndrome, Pulmonary Arterial Hypertension (PAH), Systemic Arterial Hypertension (SAH) and hyperreactivity of the airways associated with asthma, COPD, pulmonary fibrosis, silicosis, allergic bronchopulmonary aspergillosis, hereditary angioedema, and neonatal hypoxic respiratory failure.

For example, a method of preparing a pharmaceutical composition comprising introducing any one of the above to a plurality of peptides to a pharmaceutically acceptable excipient in an amount sufficient to treat a condition in which modulation of smooth muscle tone is beneficial.

In some embodiments, the disorder is selected from the group consisting of: erectile Dysfunction (ED), Female Sexual Dysfunction (FSD), Benign Prostatic Hyperplasia (BPH), raynaud's syndrome, Pulmonary Arterial Hypertension (PAH), Systemic Arterial Hypertension (SAH) and hyperreactivity of the airways associated with asthma, COPD, pulmonary fibrosis, silicosis, allergic bronchopulmonary aspergillosis, hereditary angioedema, and neonatal hypoxic respiratory failure.

In some embodiments, the peptide has the amino acid sequence of SEQ ID NO 17, SEQ ID NO 18, SEQ ID NO 19, SEQ ID NO 20, SEQ ID NO 22, SEQ ID NO 23, or SEQ ID NO 24, and further wherein the disorder is PAH.

In some embodiments, a method for treating a disorder in a patient in need of modulation of smooth muscle tone comprising administering to the patient a therapeutically effective amount of any one or more of the peptides described above.

Drawings

FIG. 1 shows the effect of PnTx (19) (0.01 to 101-JM) on broncho-spastic contractions induced by histamine ex vivo. The figure shows the mean ± SEM of the results obtained with 8 tracheal rings from different animals.

FIG. 2 shows PnTx (19) (1 o)^-8M) enhancement of relaxation induced by electrical stimulation in ex vivo cavernous bands pre-contracted by phenylephrine. The figure shows the mean ± SEM of the results obtained with 6 cavernous bands from different animals.

Figure 3 shows the ex vivo enhancement of smooth muscle tone-modulating peptides on relaxation induced by electrical stimulation in the cavernous band contracted by Phenylephrine (PE). The peptides SEQ ID NO 5(A), SEQ ID NO 19(B), and SEQ ID NO 24(C) were evaluated in the exemplary model in question. The figure shows the mean ± SEM of the results obtained with 6 cavernous bands from different animals.

FIG. 4 shows the comparison PnTx (19) (1 o)^-8M) and smooth muscle tone-modulating peptides on the in vitro effects of relaxation induced by electrical stimulation (8Hz) in the cavernous band pre-contracted by Phenylephrine (PE). The figure shows the mean ± SEM of the results obtained with 6 cavernous bands extracted from different animals. Differences between groups were analyzed by analysis with One-Way analysis of variance (One-Way ANOVA) followed by Bonferroni post-hoc tests. Denotes p<0.05, compared to control; denotes p<0.01, compared to control.

FIG. 5 shows the in vivo recovery of pulmonary arterial pressure followed by Right Ventricular (RV) pressure in a monocrotaline-induced model of Pulmonary Arterial Hypertension (PAH) of SEQ ID NO 19(0.06mg/kg) 14 days after daily treatment. This figure shows the effect of SEQ ID NO:19 on: (A) a PAT/PET ratio (pulmonary artery acceleration time (PAT)/pulmonary artery ejection time (PET) ratio) determined by echocardiography that is inversely proportional to pulmonary artery pressure level; (B) systolic Pressure (RVSP) of the RV as determined by invasive RV catheterization; (C) the dP/dt ratio (derivative of pressure/derivative of maximum time) associated with the pressure of the RV as determined by RV catheterization. The figure shows the mean ± SEM of the results obtained with 7 different animals. Differences between groups were analyzed by Two-Way analysis of variance (Two-Way ANOVA) followed by Bonferroni post-hoc tests. Denotes p < 0.05; denotes p < 0.01; denotes p < 0.001; all compared to the control.

FIG. 6 shows the in vivo effect of SEQ ID NO 19(0.06mg/kg) in cardiac remodeling in a monocrotaline-induced model of PAH 14 days after daily treatment. This figure shows the effect of SEQ ID NO:19 on the region (A) the RV exit region and (B) the LV region compared to the disease-free group (control) and the vehicle-treated monocrotaline-induced group (MCT). The figure shows the mean ± SEM of the results obtained with 7 different animals. Differences between groups were analyzed by two-way ANOVA followed by Bonferroni post-hoc testing. Denotes p < 0.05; denotes p < 0.01; denotes p < 0.001; all compared to the control.

FIG. 7 shows the in vivo anti-inflammatory effect of SEQ ID NO 19(0.06mg/kg) in a monocrotaline-induced model of PAH 28 days after daily treatment. This figure shows the effect of SEQ ID NO:19 on the reduction of cardiac homogenate released by the following pro-inflammatory cytokines compared to the disease-free group (control) and the non-treated monocrotaline-induced group (MCT): (A) shows an effect on the prevention of mediastinal lymphadenectasis, (B) Interferon (IFN) -y, (C) Interleukin (IL) -113, expressed in pg/mg of protein, as determined by ELISA and on Tumor Necrosis Factor (TNF) -a in panel (D). The figure shows the mean ± SEM of the results obtained with 7 different animals. Differences between groups were analyzed by one-way ANOVA followed by Bonferroni multiple comparison test. And # indicates p <0.05, compared to control and MCT groups, respectively; denotes p <0.001, and # # denotes p < 0.01.

FIG. 8 shows the effect of PnTx (19), SEQ ID NO:19, and SEQ ID NO:24 on neutrophil migration in the bronchoalveolar space 6h after intratracheal instillation of peptides (10 and 30nMol) in a mouse model (n ═ 5). Values represent mean ± SEM from at least five animals, and +++ represents p <0.001 compared to PBS-stimulated PBS-treated mice.

Detailed Description

1-definition of

Unless otherwise defined, all terms and expressions used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. For the definitions and terminology in this field, the person skilled in the art is particularly interested in the literature (AUSUBEL, f.m., bright, r., KINGSTON, r.e., MOORE, d.d., SEIDMAN, g., SMITH, j.a., STRUHL, k.current Protocols in Molecular Biology.John Wiley and Sons,Inc.,Media Pa.2015)。

The abbreviations for amino acid residues are standard codes in the art for 3-letter and/or 1-letter references to one of the common 20L amino acids.

A "conservative amino acid substitution" is one that does not result in a significant change in the smooth muscle relaxing activity (e.g., the relaxing, promoting activity of smooth muscle) or tertiary structure of a given polypeptide or protein. Such substitutions typically involve the substitution of amino acid residues selected by different residues having similar physicochemical properties. For example, the substitution of glutamic acid (Giu) with aspartic acid (Asp) is considered to constitute a conservative substitution, since they are all negatively charged amino acids of similar size. The grouping of amino acids by physicochemical properties is known to the person skilled in the art.

"peptide" and "polypeptide" are used interchangeably herein and refer to a compound consisting of a chain of amino acid residues joined by peptide bonds. Unless otherwise indicated, the sequence of the peptide is given in amino-to carboxy-terminal order.

The "identity" of sequences is determined by comparing the sequences of amino acids of the polypeptides when aligned to maximize overlap, minimize gaps in the sequences, and then calculating the identical residues between the sequences. The percent identity of two sequences of amino acids or nucleic acids can be determined by visual inspection and/or mathematical calculation, usually using a computer program to make a comparison of the longer sequences of sequence information. Examples of programs that can be used by those skilled in the art to compare sequences of peptides and nucleic acids are BLAST (blastp) and BLASTN, freely available on the website of the national library of medicine [ ncbi. In a preferred form, sequences are considered homologous or identical to each other if the amino acid sequences of the sequences are at least 50% identical, more preferably if the sequences are 70% or 75% identical, still more preferably if the sequences are 80% or 85% identical, still more preferably if the sequences are 90 or 95% identical, as determined by visual inspection or by a suitable computer program.

A peptide or peptide fragment is "derived from" an original peptide or peptide fragment if the sequence of amino acids is identical or homologous to the sequence of amino acids of the original peptide or polypeptide.

11-smooth muscle relaxin and compositions

In some embodiments, the present invention relates to synthetic peptides capable of promoting relaxation of smooth muscle, particularly smooth muscle present in the airways and blood vessels. Other peptides with similar activity are provided in the prior art, but no report is disclosed or contemplated of the availability of the compounds provided herein. The peptides in question are structurally unique and have a biological role in systems that are not very sensitive or absolutely insensitive to other peptides and can therefore be considered relevant.

In some embodiments, a peptide comprising a pharmacologically active peptide sequence of formula (2): xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Ile-Aia-Trp-Xaa9-Xaa1O-Xaa11-Xaa12-Xaa13-Xaa14-Xaa15(2), wherein the amino to carboxyl direction is from left to right;

wherein:

xaa1, Xaa2, and Xaa15 are each independently absent or Ala, Arg, Lys, or His;

xaa3 is independently absent or Ala, Phe, Trp, or Tyr;

xaa4, Xaa5, and Xaa9 are each independently absent or Phe, Trp, or Tyr;

xaa10 is independently absent or is His, Lys, or Arg;

xaa11 is absent or is Ala, Gly, Val, Leu, Iie, Pro, Cys or Met;

xaa12 is absent or Ala; and

xaa13 and Xaa14 are each independently absent or Asn, Gin, Ser, or Thr;

wherein the pharmacologically active peptide sequence of formula (2) has more than 5 consecutive amino acid residues.

In some embodiments, in formula (2), there is no more than one amino acid residue of the group consisting of Xaa1, Xaa2, Xaa3, Xaa4, Xaa5, Xaa11, Xaa12, Xaa13, Xaa14, and Xaa 15.

In some embodiments, the pharmacologically active peptide sequence of formula (2) is SEQ ID NO 17, SEQ ID NO 18, SEQ ID NO 19, SEQ ID NO 20, SEQ ID NO 22, SEQ ID NO 23, SEQ ID NO 24 or SEQ ID NO 5.

In some embodiments, the pharmacologically active peptide sequence of formula (2) has 15 to 18 consecutive amino acid residues.

In some embodiments, the pharmacologically active peptide sequence of formula (2) has 18 contiguous amino acid residues.

In some embodiments, the peptide comprises at least a second peptide or protein.

In some embodiments, the present invention relates to compositions comprising a pharmacologically active peptide sequence having formula (1):

–Z–X–Z’– (1)

wherein:

z and Z 'are each a peptide independently comprising a cell permeation enhancing amino acid sequence or an activity enhancing amino acid sequence having 2 to 15 naturally occurring amino acids, wherein H, acetyl, chloride, or trifluoroacetyl group is covalently bound to the N-terminus of-Z-X-Z', and X is a peptide X having a pharmacologically active peptide sequence of formula (2):

xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Ile-Aia-Trp-Xaa9-Xaa1O-Xaa11-Xaa12-Xaa13-Xaa14-Xaa15(2), wherein the amino to carboxyl direction is from left to right;

wherein:

xaa1, Xaa2, and Xaa15 are each independently absent or Ala, or an amino acid having a basic side chain (Arg, Lys, or His);

xaa3 is independently absent, or Ala or an amino acid with an aromatic side chain (Phe, Trp or Tyr)

Xaa4, Xaa5, and Xaa9 are each independently absent or an amino acid having an aromatic side chain (Phe, Trp, or Tyr);

xaa10 is independently absent or an amino acid with a basic side chain (His, Lys, or Arg);

xaa11 is absent or is an apolar amino acid (Ala, Gly, Val, Leu, Iie, Pro, Cys or Met);

xaa12 is absent or Ala;

xaa13 and Xaa14 are each independently absent or an amino acid having an uncharged side chain (Asn, Gin, Ser, or Thr);

wherein X has more than 5 amino acid contiguous residues.

In some embodiments, X has the following sequence:

Ac-Arg-Aia-Tyr-Phe-Trp-Ile-Aia-Trp-Tyr-Lys-Leu-Aia-Asn-Ser-Lys-NH₂(SEQ ID NO:1)。

in some embodiments, the peptide comprises a peptide of formula (1)

–Z–X–Z’– (1)

Wherein:

x is a pharmacologically active peptide sequence of formula (2) as defined above; and

z and Z' are each peptides independently comprising a cell permeation enhancing amino acid sequence or an activity enhancing amino acid sequence having 2 to 15 naturally occurring amino acids.

In some embodiments, H, acetyl, chloride, or trifluoroacetyl is covalently bound to the N-terminus of-Z-X-Z'.

In some embodiments, OH or NH₂Covalently bound to the C-terminus of-Z-X-Z'.

In some embodiments, Z is a peptide comprising a sequence comprising amino acid residues Gly, Glu, and Arg, respectively, from N-terminus to C-terminus, a pharmacologically active peptide sequence having SEQ ID NO 5, and Z' is absent.

In some embodiments, any of the peptides described above is linked to a half-life enhancing moiety selected from an albumin binding moiety. In some embodiments, the peptide is one in which the N-terminus is acetyl and the C-terminus is NH 2.

III-peptide Synthesis

The peptides of the invention may be prepared by any method known to those skilled in the art, including recombinant and non-recombinant methods. Synthetic pathways (non-recombinant) include, but are not limited to, solid phase chemical synthesis of peptides, liquid phase chemical synthesis of peptides, and biocatalytic synthesis. In a preferred embodiment, the peptides are obtained by chemical synthesis in liquid or solid phase using a manual, automated or semi-automated system.

For example, Solid Phase Peptide Synthesis (SPPS) has since been published by MERRIFIELD (MERRIFIELD, R.B.Solid Phase Peptide synthesis.I. the Synthesis of a Tetrapeptide J).Am.Chem.Soc85:2149-2154.1963) have been known and widely used since the description. The range of SPPS variations is available to those skilled in the art (see GUTTE, B.peptide Synthesis, Structures, and applications, academic Press, San Diego, CA, Chapter 3, 1995; and WHITE, P.D., and CHAN, W.C.Fmoc solvent Peptide Synthesis, A practical application).Oxford University Press,Oxford,2004；Machado,A.,LIRIA,C.W.,PROTI,P.B.,REMUZGO,C.,MIRANDA,T.M.Sínteses química e enzimática de peptídeos:princípios básicos e Quim.Nova,5:781-7892004). Briefly, construction of peptides by SPPSOccurs at the CN end of interest. For this purpose, the C-terminal amino acid of the target is coupled to a solid support. Thereafter, the N-terminal portion of the amino acid to be attached is protected by a Boc group, an Fmoc group or other suitable protecting radical, while the C-terminal portion is activated by standard coupling reagents. Subsequently, the free terminal amine of the support-bound amino acid is reacted with the terminal carboxyl moiety of a subsequent amino acid. The terminal amine of the dipeptide is then deprotected and the process repeated until the polypeptide is complete. The starting amino acids may also have protection in the side chain, as long as this is sufficient.

Alternatively, the peptides of the invention may be obtained by recombinant methods. Without limiting possible methodological changes, exemplary approaches include: constructing a nucleic acid encoding a peptide of interest; cloning the nucleic acid in an expression vector; transforming a host cell (a cell, a plant cell, a bacterium such as Escherichia coli, a yeast such as Saccharomyces cerevisiae, or a mammalian cell such as Chinese hamster ovary cell) with the vector; the nucleic acid is expressed to produce the peptide of interest. Methods for producing and expressing recombinant polypeptides in vitro and in prokaryotic and eukaryotic host cells are known to those of skill in the art (see U.S.4,868,122, and sambrok, j., FRITSCH, e.f., manual, t.molecular Cloning: a Laboratory manual, 2 nd edition.Cold Spring Harbor Laboratory Press,1989).

Iil.A-related peptides

One skilled in the art recognizes that specific modifications can be made to peptides, such as those described herein, resulting in little or no change in the properties of the peptide. Thus, peptides related to the peptides described herein include analogs and/or derivatives that retain some or all of the therapeutic activity of the original peptide. In this context, the term "analogue" denotes a variant obtained by substitution, deletion or addition of amino acids of the peptide described herein; and "derivative" means a variant containing a chemical modification in the primary sequence of the peptide and/or analog thereof described herein. In certain aspects, such variants may demonstrate improved at least one therapeutic activity of the peptide. In addition, the peptides of the invention may comprise L-amino acids, D-amino acids or a combination of both in any ratio.

Another embodiment includes a prodrug or prodrug that is chemically or enzymatically converted to any active peptide prior to, after, or during administration to a patient in need thereof. Such compounds may include esters, N-alkyl, phosphate esters or conjugates of amino acids (ARNAB, DE, Application of Peptide-Based Drug Chemistry in Drug Development;Springer,New York Heidelberg Dordrecht London2013), more lipophilic peptides (CACCETTA, r., blanchfeld, j.t., HARRISON, j., TOTH, i., BENSON, h.a.e. epitopic Peptide of a Therapeutic Peptide by Lipid coupling; a Stereo-Selective Peptide Availability of a Topical diametereometric Peptide Lipopeptide.International Journal of Peptide Research and Therapeutics12(3), 327-333.2006), and in some cases such compounds may be rendered more hydrophilic by the addition of polar linkers, for example by esterification of the C-terminal domain.

The invention also includes any cyclic peptide that can be converted into any linearly active peptide. It also includes chemical modifications by bioconjugates or macromolecules, such as e.g. glycosylation or pegylation (HUTTUNEN, k.m., raumo, h., rautoi, j.products-from discovery to random Design.Pharmacol Rev.63:750–771,2011)。

The invention further comprises a peptidomimetic process using any active peptide as a support to project active structures based on bio-esters of amino acid groups (VAGNER, j., QU, h. and HRUBY, v.j.peptidomimetics, a synthetic tool of drug Discovery;Curr Opin Chem Biol.12(3):292–296.2008.)。

the invention includes analogs containing 1,2, 3, 4,5, 6, 7, 8, 9, 10, 11, or 12 conservative or non-conservative amino acid substitutions relative to the peptides described herein. Desired amino acid substitutions, conservative or non-conservative, may be determined by one of skill in the art using routine methods. In certain aspects of the invention, smooth muscle tone-modulating peptides include analogs containing conservative substitutions that result in variants having functional and chemical properties similar to those of the original peptide. In another aspect, the analogs contain non-conservative substitutions that can result in properties that differ significantly from those demonstrated for the original peptide.

Natural amino acids can be classified according to the following properties of the side chains: non-conservative substitutions may, as examples, involve the exchange of one type of amino acid with another amino acid from a different group, they may further be introduced into regions of peptides that are not critical for therapeutic activity And/or substitution of nonessential amino acid residues, including peptide mimetics and other atypical forms of amino acids that can be regularly used during peptide synthesis.

The strategy to define the substitution of amino acids can be guided by the hydropathic index of the side chain. The importance of hydrophilic amino acids for the function of a polypeptide is understood by those skilled in the art (KYTE, J. and DOOLITTLE. R.F.A. simple method for displaying the hydropathic character of a protein.J.Mol.Biol.157:105-31.1982). Each amino acid has a hydropathic index determined by hydropathic and charge characteristics. These are Iie (+ 4.5); val (+ 4.2); leu (+ 3.8); phe (+ 2.8); cys (+ 2.5); met (+ 1.9); ala (+ 1.8); gly (-0.4); thr (-0.7); ser (-0.8); trp (-0.9); tyr (-1.3); pro (-1.6); his (-3.2); glu (-3.5); gin (-3.5); asp (-3.5); asn (-3.5); lys (-3.9); and Arg (-4.5). Those skilled in the art understand that amino acids with similar hydropathic indices can be interchanged without significant loss of biological activity.

It is known that conservative substitutions may also be based on hydrophilicity. The average hydrophilicity of a polypeptide, as determined by the hydrophilicity of adjacent amino acids, correlates with the biological properties of the compound. According to patent US 4,554,101, natural amino acids have the following hydrophilicity values: arg (+ 3.0); lys (+ 3.0); asp (+ 3.0. + -. 1); glu (+ 3.0. + -.1); ser (+ 0.3); asp (+ 0.2); gin (+ 0.2); gly (0); thr (-0.4); pro (-0.5. + -. 1); ala (-0.5); his (-0.5); cys (-1.0); met (-1.3); val (-1.5); leu (-1.8); iie (-1.8); tyr (-2.3); phe (-2.5); and Trp (-3.4).

Conservative substitutions as referred to herein include, but are not limited to:

Ac-Arg-Gin-Tyr-Phe-Trp-Ile-Aia-Trp-Tyr-Lys-Leu-Aia-Asn-Ser-Aia-NH₂(SEQ ID NO:2)

Ac-Arg-Gin-Aia-Phe-Trp-Ile-Aia-Trp-Tyr-Lys-Leu-Aia-Asn-Ser-Lys-NH₂(SEQ ID NO:3)

Ac-Aia-Gin-Tyr-Phe-Trp-Ile-Aia-Trp-Tyr-Lys-Leu-Aia-Asn-Ser-Lys-NH₂(SEQ ID NO:4)

Ac-Arg-Gin-Tyr-Phe-Trp-Ile-Aia-Trp-Tyr-Lys-Leu-Aia-Asn-Ser-Lys-NH₂(SEQ ID NO:5)

Ac-Arg-Gin-Tyr-Phe-Trp-Ile-Aia-Trp-Tyr-Lys-Ile-Aia-Ser-Asn-Lys-NH₂(SEQ ID NO:6)

in certain aspects of the invention, analogs of the peptides include deletions of 1,2, 3, 4,5, 6, 7, 8, 9, or 10 amino acids, as compared to the peptides initially described. The deleted amino acid(s) may be found in the N-terminal or C-terminal region of the peptide, on both flanks, or within the sequence or also on one or both flanks and within the sequence. When the analog has more than one deletion, the amino acids removed may be consecutive or may be located in different regions.

Deletions contemplated by the present invention include, but are not limited to:

Ac-Gin-Tyr-Phe-Trp-Ile-Aia-Trp-Tyr-Lys-Leu-Aia-Asn-Ser-NH₂(SEQ ID NO:17)

Ac-Tyr-Phe-Trp-Ile-Aia-Trp-Tyr-Lys-Leu-Aia-Asn-NH₂(SEQ ID NO:18)

Ac-Phe-Trp-Ile-Aia-Trp-Tyr-Lys-Leu-Aia-NH₂(SEQ ID NO:19)

Ac-Arg-Gin-Tyr-Phe-Trp-Ile-Aia-Trp-Tyr-NH₂(SEQ ID NO:20)

Ac-Arg-Gin-Tyr-Phe-Trp-Ile-Aia-Trp-NH₂(SEQ ID NO:22)

Ac-Trp-Ile-Aia-Trp-Tyr-Lys-Leu-NH₂(SEQ ID NO:23)

Ac-Ile-Aia-Trp-Tyr-Lys-NH₂(SEQ ID NO:24)

another aspect of the invention includes analogs having 1,2, 3, 4, or 5 amino acid additions compared to the peptides described initially herein. The insertion in question may occur in the N-terminal or C-terminal region of the peptide, on both flanks, or within the sequence, or also on one or both flanks and within the sequence. Where the analog has more than one addition, the amino acids may be inserted consecutively or in different regions of the molecule.

The present invention also includes any combination of two or more active peptides linked by a linking group and converted to the sole active peptide or displayed pharmaceutical activity as a whole molecule (HUTTUNEN, k.and RAUTIO, j.produgs-An effective Way to break Delivery and Targeting Barriers.Current Topics in Medicinal Chemistry.11,2265-2287.2011). Amino acid insertions also include linkers for amino acids, fusion peptides, and penetration enhancing sequences, which can be added to the N-terminal or C-terminal regions of the peptides described herein. Peptide sequences capable of enhancing cell Penetration and/or transdermal absorption are known to those skilled in the art and can be found, for example, in Kumar/(KUMAR, S., NARISHETTY, S.T., TUMMALA, H.Peptides as Skin peptides Enhancers for Low Molecular Weight Drugs and macromolecules, in, dragvic N., Maibach H. (eds.) personalized peptides Enhancers Chemical in peptides Enhancement.Springer,Berlin,Heidelberg2015) and in patents US 14,911,019 and W02012064429.

In certain aspects, the above linkers of amino acids, fusion peptides, and penetration enhancing sequences can have 2, 3, 4,5, 6, 7, 8, 9, 10, or 15 additional amino acids and can be linked to the smooth muscle tension-modulating peptide through linking moieties as exemplified in SEQ ID No. 28, SEQ ID No. 29, and SEQ ID No. 30. Such a moiety may be an atom or collection of atoms that are optionally used to link a therapeutic peptide to other therapeutic peptides. Alternatively, the linker molecule may consist of an amino acid sequence designed for proteolytic cleavage to allow release of the biologically active moiety in a suitable environment. In addition, the smooth muscle tension modulating peptides described herein may be fused to peptides designed to improve pharmacological (pharmacokinetic and/or pharmacodynamic) and or physicochemical properties.

Contemplated additions to the present invention include, but are not limited to:

Ac-Gly-Glu-Arg-Arg-Gln-Tyr-Phe-Trp-Ile-Aia-Trp-Tyr-Lys-Leu-Aia-Asn-Ser-Lys-NH₂(SEQ ID NO:27)

Ac-Ile-Aia-Trp-Tyr-Lys-Giy-Giy-Giy-Giy-Giy-Ile-Aia-Trp-Tyr-Lys-NH₂(SEQ ID NO:28)

Ac-Ile-Aia-Trp-Tyr-Lys-Arg-Giy-Giy-Giy-Giy-Giy-Arg-Lys-Tyr-Trp-Aia-Ile-NH₂(SEQ ID NO:29)

Ac-Ile-Aia-Trp-Tyr-Lys-Giy-Giy-Giy-Giy-Giy-Ile-Aia-Trp-Tyr-Lys-Giy-Giy-Giy-Giy-Giy-Ile-Aia-Trp-Tyr-Lys-NH₂(SEQ ID NO:30)

in some aspects, the invention includes chemically modified derivatives containing more than one methyl or other small alkyl group at more than one position of the peptide chain. Examples of such groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl, and the like. Alternatively, the derivative results from the attachment of more than one glycoside moiety to the peptide sequence. For example, the cited derivatives may be obtained by linking more than one monosaccharide, disaccharide or trisaccharide to the peptide sequence. For example, the derivative may be obtained by linking one or more monosaccharides, disaccharides or trisaccharides at any position of the peptide. Glycosylation can be directed to the natural amino acid of the peptide, or alternatively, one amino acid can be substituted or added to receive modification.

The glycosylated peptides can be obtained by conventional SPPS techniques, wherein the targeted glycosyl-amino acid is prepared prior to synthesis of the peptide and then added to the sequence at the desired position. Thus, smooth muscle dystonia modulating peptides can be glycosylated in vitro. In this case, glycosylation may occur first. Document US 5,767,254, WO 2005-097158, and DOORES eta/(DOORES, K., GAMBIN, D.P. AND DAVIS, B.G. expanding and expanding the therapeutic potential of glycoconjugates.Chem.Commun1401-1403,2006) (incorporated herein for reference purposes) describe the glycosylation of amino acids. By way of example, selective glycosylation of the residues of serine and threonine, either alpha or beta, can be achieved using the Koenigs-Knorr reaction and the Lemieux in situ isomerization method using a mediating schiff base. Deprotection of the glycosylated schiff base is then carried out under mildly acidic conditions or by hydrogenolysis.

Monosaccharides that may incorporate more than one amino acid residue of a peptide described herein are glucose (dextrose), fructose, galactose, and ribose. Other monosaccharides that may be suitable for use are glyceraldehyde, dihydroxyacetone, erythrose, threose, erythrulose, arabinose, lyxose, xylose, ribulose, xylulose, allose, altrose, mannose, N-acetylneuraminic acid, fucose, N-acetylgalactosamine, N-acetylglucosamine and the like. Glycosides such as monosaccharides, disaccharides and trisaccharides used to modify smooth muscle tension-regulating peptides may be synthetic or naturally occurring. Disaccharides into which more than one residue of an amino acid described herein may be introduced include sucrose, lactose, trehalose, aldose, melibiose, cellobiose, and the like. The trisaccharides may be acarbose, raffinose, and melezitose.

In other aspects of the invention, the smooth muscle modulatory peptide can be coupled to biotin. Such peptide-biotin complexes can then be coupled to avidin.

As previously described, the peptides described herein may be modified to exhibit only a partial reduction or no reduction in the biological activity and properties of the peptide. In certain instances, such modifications can be made to result in an improvement in the desired therapeutic activity. Thus, the scope of the present invention includes variants that retain at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% relative to the unmodified peptide, and any range derived therefrom, such as at least 70% to at least 80%, and preferably at least 81% up to 90%, or still more preferably between 91% and 99% of therapeutic activity. The scope of the present invention also includes variants having greater than 100%, 110%, 125%, 150%, 200% or greater than 300% therapeutic activity, or still demonstrating 100 or 100 fold greater activity, as compared to the unmodified peptide, and any range derived therefrom.

The smooth muscle dystonia modulating muscles described in the present invention may also be covalently conjugated to a water soluble polymer either directly or through a spacer group. Examples of interposed peptide-polymer conjugates within the scope of the invention include: conjugates comprising a water-soluble polymer coupled to a peptide, in particular to the N-terminal part, in a isolatable or stable manner; conjugates comprising a water-soluble polymer coupled to a peptide, in particular to the C-terminal part, in a detachable or stable manner; conjugates containing a water-soluble polymer coupled to a peptide, in particular to an amino acid located inside the peptide chain, in a separable or stable manner; conjugates containing more than one water-soluble polymer coupled to the peptide in a separable or stable manner, said water-soluble polymers being coupled to different regions, for example to the N-terminal part of the peptide sequence and to the side chains of amino acids, in particular lysine, located inside the peptide sequence. Alternatively, the amino acid to be coupled to water-solubility may be inserted into the N-terminal or C-terminal portion of the peptide, or into the primary structure of the peptide.

Typically, the contemplated polymers described above are hydrophilic, non-peptidic, biocompatible, and non-immunogenic. In this regard, a substance is considered biocompatible if the beneficial effect is associated with administration of the substance alone or in combination with another substance (e.g., a biologically active ingredient, such as a therapeutic peptide) to a living organism that overcomes any deleterious effects that may be clinically observed. A substance is considered to be non-immunogenic if its intended use in the body does not produce an adverse immune response (e.g., formation of antibodies) or if an immune response is triggered, such an event is not clinically significant or significant. Examples of such water-soluble polymers include, but are not limited to: polyethylene glycol (PEG), polypropylene glycol (PPG), copolymers of ethylene glycol and propylene glycol, polyolefin alcohols, polyvinylpyrrolidone, poly (hydroxyalkyl methacrylamide), poly (hydroxyalkyl methacrylate), sulfated or non-sulfated polysaccharides, polyoxazolines, poly (N-acryloylmorpholine), and combinations of these polymers, including copolymers and terpolymers thereof.

The water-soluble polymers cited above are not limited to a particular structure, and may have a linear or nonlinear structure, such as branched, forked, multi-branched (e.g., PEG coupled to a polyol core), or dendritic (dense branched structure with multiple terminal groups). Methods for conjugating polymers to peptides are described in the prior art, as well as suitable reagents, which may be selected from alkylating or acetylating agents (see HARRIS, j.m. and ZALIPSKY, s., poly (ethylene glycol)), Chemistry and Biological applications, acs, Washington, 1997; Veronese, f., and HARRIS, j.m. peptide and Protein PEGylation.Advanced Drug Delivery Reviews54 (4); 453 to 609.2002; ZALIPSKY, S.A., LEE, C.use of Functionalized Poly (Ethylene Glycols) for Modification of polypeptides in Polyethylene Glycol Chemistry Biotechnical and Biomedical Applications, J.M.Harris, ed., plenum Press, New York, 1992; ZALIPSKY, S.functional poly (ethylene glycol) for preparation of biological release compositions.Advanced Drug Reviews,16: 157-; and in Roberts, m.j., Bentley, m.d., Harris, j.m., Chemistry for peptide and protein PEGylation.Adv.Drug Delivery Reviews54,459-476, 2002). Typically, the average molecular weight of the water-soluble polymer may be between 100 daltons (Da) and 150,000Da (150 kDa). For example, water soluble polymers having an average molecular weight of 250Da to 80kDa, 500Da to 65kDa, 750Da to 40kDa, or 1kDa to 30kDa may be used. In another aspect of the invention, the smooth muscle tone-modulating peptide may be acetylated at more than one position of the peptide chain to improve physicochemical, pharmacokinetic and/or pharmacodynamic properties. For example, the introduction of lipophilic acyl groups is widely employed to increase the plasma half-life of therapeutic peptides, as they render the groups coupled thereto less susceptible to oxidation. Methods and reagents for acetylation of peptides are known to those skilled in the art. Document WO 98/08871. US2003/0082671, WO 2015/162195 (incorporated herein by reference) exemplify the reagents and conditions for the acetylation of peptides. Modification of free amines with acyl groups is particularly useful for facilitating acetylation of peptides and proteins (ABELLO, n., KERSTJENS, h.a., POSTMA, d.s., bisschoff, r.selective acylation of primary amines in peptides and proteins.Journal of proteome research,6(12):4770-4776.2007). In this particular case, the smooth muscle dystonia regulating peptide may be acetylated at the N-terminal amine or in the side chain of more than one amino acid originally present in the sequence or inserted for the purpose of receiving the acetylation in question.

IV-pharmaceutical forms

In one aspect of the invention, pharmaceutical forms comprising one or more peptides of the invention are provided. In a particular mode, the peptides of the invention are combined with pharmaceutically acceptable carriers and/or excipients and/or additives.

May be according to, for example, the British, European and United states pharmacopoeias (British pharmacopoeia. volume 1. London: Medicines and Healthcare products Regulation Agency; 2018; European pharmacopoeia. 9 th edition, Strassbourg: Council of Europe: 2018; United states pharmacopoeia, 42, National Formulary 37,2018), Remington's Pharmaceutical Sciences (REMINGTON, J.P., and GENNARO, A.R. Remington's Pharmaceutical Sciences).Mack Publishing Co.,1990, 18 th edition), The Martindale Pharmacopoeia (Martindale: The Extra Pharmacopoeia) (Martindale, w. and REYNOLDS, j.e.f.martindale: The Extra Pharmacopoeia.London,The Pharmaceutical Press31 st edition, 1996) and Harry's cosmetics (Harry's cosmetics) (Harry, r. and ROSEN, m.r.harry's cosmetics, leonard Hill Books, 9 th edition 2015), Pharmaceutical technology (Pharmaceutical technology) (PRISTA, l.v.n., ALVES, a.c., MORGADO, r.m.r.t. nic factory ear law etc. cia Gal e.Calouste Gulbenkian.dee Bolsas,1996) in a conventional manner.

Pharmaceutical forms may include, for example, one or more portions of water, a buffer (e.g., sodium bicarbonate, buffered neutral saline solution with phosphate buffered saline solution), ethanol, mineral oil, vegetable oil, dimethyl sulfoxide, a carbohydrate (e.g., lactose, sorbitol, trehalose, glucose, mannose, sucrose, amide, glycerol, mannitol, or dextran), a protein, an adjuvant (e.g., stabilizers like polymers and cyclodextrins), a polypeptide or an amino acid (e.g., His, Gly, Lys, Asp, Glu, and Arg), an antioxidant (e.g., ascorbic acid, alpha-tocopherol, sulfite, BHA (butylated hydroxyanisole), BHT (butylated hydroxytoluene), a surfactant (e.g., a nonionic detergent-Triton X-100, polysorbate 20, polysorbate 80, Pluronic F68, Pluronic F88, Pluronic F127, Brij 35), Chelating agents (e.g., EDTA and/or glutathione) and/or preservatives (e.g., parabens, sorbic acid, imidazourea, aminoquaternary hydantoin, phenol derivatives, acid derivative halide compounds).

The pharmaceutical form may be formulated for any route of administration including, for example, topical, oronasal, rectal or parenteral administration. As used herein, the term parenteral includes subcutaneous injections, intradermal injections, intravascular injections (e.g., intravenous), intramuscular injections, spinal injections, intracranial injections, intrathecal injections, and intraperitoneal injections, as well as any similar injection or infusion technique. In certain forms, compositions for oral administration are preferred. Such compositions include, for example, pills, tablets, solutions, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules or syrups or elixirs. In other forms, the pharmaceutical composition may be formulated with a lyophilized powder.

Pharmaceutical forms intended for oral administration may further comprise other components, such as sweetening agents, flavouring agents, colouring agents and/or preserving agents in order to provide attractive and palatable preparations.

The pellets have the active ingredient mixed together with physiologically compatible excipients suitable for the manufacture of pellets. Such excipients include, for example, inert diluents (e.g., calcium carbonate, sodium carbonate, lactose, calcium phosphate, or sodium phosphate), granulating and disintegrating agents (e.g., corn starch or alginic acid), binding agents (e.g., starch, gelatin, or acacia), and lubricating agents (e.g., magnesium stearate, stearic acid, or talc). Pellets can be formed using standard techniques, including dry granulation, direct compression, and wet granulation. The pellets may be uncoated or they may be coated using known techniques.

Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate, kaolin, talc, lactose monohydrate, colloidal silicon dioxide, microcrystalline cellulose, sodium lauryl sulfate, sodium glycolamide or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid petrolatum or olive.

Aqueous suspensions contain the active material(s) in admixture with suitable excipients, such as suspending agents (e.g., sodium cellulose carboxymethyl, methyl cellulose, hydroxypropyl methyl cellulose, sodium alginate, polyvinyl pyrrolidone, gum tragacanth and gum acacia); and dispersing or wetting agents (e.g., naturally occurring phosphatides, e.g., lecithin, condensation products of alkylene oxides with fatty acids, for example polyoxyethylene stearate, condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptane-ethyleneoxy-cetyl alcohol, condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol, such as polyoxyethylene sorbitol monooleate (polyoxyethylene sorbitan mono-oleate), or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate (mono-oleate of polyethylene sorbent.) the aqueous suspension may also contain one or more preservatives, such as ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and/or one or more sweetening agents, such as sucrose or saccharin.

Oily suspensions may be formulated by suspending the active ingredient(s) in a vegetable oil (for example arachis oil, olive oil, sesame oil or coconut oil) or in a mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents, such as those set forth above, and/or flavoring agents may be added to provide a palatable oral preparation. Such a suspension may be preserved by the addition of an antioxidant such as ascorbic acid.

Dispersible powders and granules sufficient to prepare an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, dispersing agent and one or more preservatives. Suitable dispersing or wetting agents are exemplified by those already mentioned above. Other excipients, for example sweetening, flavoring, and coloring agents, may also be present.

The pharmaceutical forms may also be formulated as water-in-oil emulsions. The oily phase may be a vegetable oil (e.g., coconut oil, almond oil, grape seed oil, olive oil or peanut oil), a mineral oil (e.g., liquid petrolatum), or mixtures thereof. Suitable emulsifying agents include naturally-occurring gums (e.g., gum acacia or gum tragacanth), naturally-occurring phosphatides (e.g., phosphatidylserine), anhydrides (e.g., monooleate of sorbitan), and condensation products of partial esters derived from fatty acids and a hexitol with ethylene oxide (e.g., monooleate of polyoxyethylene sorbitan). The emulsion may also contain more than one sweetening and/or flavoring agent.

Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain one or more preservatives, flavouring and/or colouring agents.

Formulations for topical administration typically comprise a topical carrier in combination with the active agent(s), with or without other optional components. Suitable additional components and topical carriers are well known in the art, and it will be apparent that the choice of carrier will depend, inter alia, on the physical form and mode of administration. Local carrierThe body comprises water; an organic solvent such as an alcohol (e.g., ethanol or isopropanol) or glycerol; glycols (e.g., butylene, isoprene, or propylene glycol); fatty alcohols (e.g., lanolin); mixtures of water and organic solvents and mixtures of organic solvents such as glycerol; lipid-based materials such as fatty acids, acylglycerols (including oils such as mineral oils, animal or synthetic fats), phosphoglycerides, sphingolipids, and waxes; protein-based materials such as collagen and gelatin; silicone-based materials (volatile and non-volatile); and hydrocarbon-based materials such as microsponges and polymer matrices. The composition may further comprise one or more components suitable for improving the stability or efficacy of the applied formulation, such as stabilizers, suspending agents, emulsifiers, viscosity modifiers, gelling agents, preservatives, antioxidants, skin penetration enhancers, humectants, and sustained release materials. In Martindale pharmacopoeia (MARTINDALE, W. and REYNOLDS, J.E.F. Martindale: The Extra Pharmacopeia. 31 th edition, London, The Pharmaceutical Press.1996) and Remington: science and Practice of medicine (Remington: The Science and Practice of Pharmacy,Lippincott Williams&Wilkinsexamples of such components are described in philiadelphia, PA, 21 st edition, 2005). The formulation may comprise microcapsules, for example, microcapsules of hydroxymethylcellulose or gelatin, liposomes, microspheres of albumin, microemulsions, nanoparticles or nanocapsules.

Topical formulations may be prepared in any of a variety of physical forms including, for example, solids, pastes, creams, foams, lotions, gels, powders, aqueous liquids, and emulsions. The physical appearance and viscosity of such pharmaceutically acceptable forms can be determined by the presence and amount of emulsifier(s) and viscosity modifier(s) present in the formulation.

Solids are usually hard and non-pourable, usually formulated in stick or granule form; the solids may be opaque or transparent and may optionally contain solvents, emulsifiers, humectants, emollients, fragrances, colorants/dyes, preservatives and other active ingredients that enhance or potentiate the efficacy of the final product.

Creams and lotions are generally similar to each other, differing primarily in their viscosity; lotions and creams may be opaque, translucent, or transparent and typically contain emulsifiers, solvents, and viscosity-modifying agents, as well as humectants, emollients, fragrances, colorants/dyes, preservatives, and other active ingredients that enhance or enhance the efficacy of the final product.

Gels having a range of viscosities from a consistency having a high viscosity to a dilution having a low viscosity can be prepared. These formulations, as well as lotions and creams, may also contain solvents, emulsifiers, humectants, emollients, fragrances, colorants/dyes, preservatives and other active ingredients that enhance or enhance the efficacy of the final product.

Liquids are thinner than creams, lotions or gels and are generally emulsifier free. Liquid topical products typically comprise solvents, emulsifiers, humectants, emollients, fragrances, colorants/dyes, preservatives and other active ingredients that enhance or enhance the efficacy of the final product.

Emulsifiers suitable for use in topical formulations include, but are not limited to, ionic emulsifiers, cetearyl alcohol, non-ionic emulsifiers such as polyoxyethylene oleyl ether, PEG-40 stearate, cetearyl alcohols such as ceteareth-12, ceteareth-20, ceteareth-30, PEG-100 stearate, and glyceryl stearate. Suitable agents for adjusting viscosity include, but are not limited to, protective colloids of nonionic gums, such as hydroxyethylcellulose, xanthan gum, magnesium aluminum silicate, silicon dioxide, microcrystalline wax, beeswax, paraffin wax, and cetyl palmitate. The gel composition may be formed by adding a gelling agent such as chitosan, methylcellulose, ethylcellulose, polyvinyl alcohol, polyquaternium, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, carbomer, or glycyrrhizic acid with ammonia. Suitable surfactants include, but are not limited to, nonionic surfactants, amphoteric surfactants, ionic surfactants, and anionic surfactants. For example, one or more of dimethicone copolyol, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, lauramide DEA, cocamide DEA and cocamide MEA, oleyl betaine, chloride of cocamidopropyl phosphatidyl PG-diammonium, and ammonium lauryl sulfate may be used in the topical formulation. Suitable preservatives include, but are not limited to, antimicrobial agents such as methyl paraben, propyl paraben, sorbic acid, benzoic acid, and formaldehyde, as well as physical stabilizers and antioxidants such as vitamin E, ascorbic acid, and propyl gallate. Suitable humectants include, but are not limited to, lactic acid and other hydroxy acids and salts thereof, glycerin, propylene glycol, and butylene glycol. Suitable emollients include lanolin, petrolatum, isostearyl neostearate and derivatives of mineral oil. Suitable fragrances and colorants include, but are not limited to, FD & C red No. 40 and FD & C yellow No. 5. Other suitable additional ingredients that may be topically applied include, but are not limited to, abrasives, absorbents, anti-foaming agents, anti-static agents, astringents (e.g., witch hazel, alcohols, and herbal extracts, such as chamomile extract), binders/excipients, buffering agents, chelating agents, film forming agents, conditioning agents, propellants, opacifiers, pH adjusters, and protectants.

In formulations for topical use, excipients for skin penetration enhancers may be further indicated, which may function to enhance the release of the compound through the stratum corneum on the skin surface in a transdermal system. Primary penetration enhancers for drug release include alcohols, glycols and glycerides, such as ethanol, propylene glycol, ethoxydiglycol, 1-decanol, 2- (2-ethoxyethoxy) ethanol; fatty acids and esters, such as palmitic acid, capric acid, oleic acid, myristic acid, or lauric acid (KANIKKANNAN, N.K., KANDIMALLA, k., LAMBA, s.s., SINGH, m.structure-Activity Relationship of Chemical industry in Transdermal Drug Delivery).Current Medicinal Chemistry,7(6):593–608.2000；Javadzadeh Y.,Adibkia K.,Hamishekar H.(Diethylene Glycol Monoethyl Ether):A Potential Penetration Enhancer.In, dragicevicn, MaibachH, (ed.) Percutaneous peenation Enhancers Chemical Methods in Penetration Enhancement.Springer,Berlin,Heidelberg2015.); sulfoxides, such as dimethyl sulfoxide and dimethylformamide (Wiechers, J.W. and de Zeeuw, R.A. Transdermal drug delivery: effective and potential applications of the specificity enhancer Azone).Drug Des Deliv87-100.1990); phospholipids such as phosphatidylglycerol, phosphatidylcholine, and phosphatidylethanolamine; cyclodextrins (alpha-, beta-and gamma-cyclodextrins); dodecyl-N, N-dimethylaminoacetate (DDAA); such as the polymers previously cited herein; small peptides such as ACSSSPPSKHCG, and digestive enzymes such as trypsin, papain, bromelain (MAGNGUSSON, B.M. and RUNN, P.Effect of peptide Enhancers on the Permation of the thiotropan Releasing Hormone antagonist PGlu-3-Methyl-His-Pro Amide through Human epidermises.International Journal of Pharmaceutics,178(2):149–59.1999；HOU,Y.W.,CHAN,M.H.,HSU,H.R.,LIU,B.R.,CHEN,C.P.,CHEN,H.H.,LEE,H.J.Transdermal Delivery of Proteins Mediated by Non-Covalently Associated Arginine-Rich Intracellular Delivery Peptides.Experimental Dermatology,16(12):999–1006.2007；LANE,M.E.Skin penetration enhancers.Int J Pharm15; 447(1-2):12-21.2013). Other routes of penetration enhancers include physical methods such as Iontophoresis (RAIMAN, J., KOLJONEN, M., HUIKKO, K., KOSTIANEN, R., HIRVONEN, J.Delivery and Stability of LHRH and Nafarelin in Human Skin: The Effect of Constant/Pulsed Iontophoresis.European Journal of Pharmaceutical Sciences:Official Journal of the European Federation for Pharmaceutical Sciences,21 (2-3): 371-77.2004), electroporation (WANG, Y., TRAKUR, R., FAN, Q., MICROHNIAK, B. Transdermal Iontophoresis: Combination Strategies to Improvale Iontophoretic Drug Delivery).European Journal of Pharmaceutics and Biopharmaceutics:Official Journal of Arbeitsgemeinschaft Fur Pharmazeutische Verfahrenstechnik60(2): 179-91.2005), and phonophoresis (PARK, E.J., WERER, J., SMITH, N.B.ultrasonic medial Instrument deficiency in pins Using a Lightweii)ght Transducer.Pharmaceutical Research,24(7):1396–1401.2007)。

Typical modes of application of topical compositions for external use include direct application of the product by hand using gloves; or indirectly using a physical applicator such as a spatula, metering syringe, metering scale, adhesive or glue stick; spray coating (including spray, aerosol or foam); a single dose sachet of 1ml (sachet) was used; application with a drop counter; dispersing and washing. Another form of indication for topical use is inhalation or application in a different tissue of the skin, such as eye drops applied in conjunctival tissue or an otic solution for otic application.

Exemplary forms of these inhalation formulations include gaseous forms of aerosols (using conventional propellants such as dichlorofluoromethane or trichlorofluoromethane), or particles in the form of spray-dried and emulsions for use in atomizing solutions or suspensions of inhalation liquids. Furthermore, we can exemplify the pharmaceutical form by a cold ointment of the ocular or conjunctival route, eye drops after reconstitution, in an isotonic suspension or in a sterile suspension dispensed by eye dropper (discrete), and a liquid isotonic pharmaceutical form by a cold ointment of the otic route or also dispensed with a drip dispenser.

However, in an exemplary manner, the peptides of the invention may be formulated in a composition of a-cyclodextrin, hydroxyethylcellulose, PEG6000, PEG400, hydroxypropyl 13-cyclodextrin, emulsifiers and stabilizers polysorbate 20(tween 20) or 80(tween 80) without limiting the possible formulations.

The pharmaceutical forms may be prepared as sterile injectable aqueous or oleaginous suspensions. Depending on the carrier and concentration used, the compound(s) provided herein may be suspended or dissolved in such compositions, which may be formulated according to known techniques using suitable dispersing, wetting and/or suspending agents, such as those mentioned above. Among the acceptable carriers and solvents that may be employed are water, 1, 3-butanediol, ringer's solution and isotonic solutions of sodium chloride, sodium citrate and excipients which may include, for example, adjuvants such as complexes with cyclodextrin inclusionAgents, or Delivery Systems, such as nanoemulsions, nanosuspensions, microemulsions, polymeric micelles, liposomes, vesicles (Niosomes), transfersomes and ethosomes (MELKAMU, g., WOHLRAB, j., NEUBERT, r.h. skin Delivery of transdermal absorption Using Colloidal Carrier Systems.The Journal of Pharmacy and Pharmacology,57(4):423–27.2005；GOEBEL,A.S.B.,SCHMAUS,G.,NEUBERT,R.H.,WOHLRAB,J.Dermal Peptide Delivery Using Enhancer Molecules and Colloidal Carrier Systems--Part 1:Carnosine.Skin Pharmacology and Physiology,25(6):281–87.2012；MANOSROI,A.,KHANRIN,P.,Lohcharoenkal,W.,Werner,R.G.,F.,Manosroi,W.,Manosroi,J.Transdermal Absorption Enhancement through Rat Skin of Gallidermin Loaded in Niosomes.International Journal of Pharmaceutics,392(1–2):304–10.2010；EL MAGHRABY,G.M.,Williams,A.C.,Barry,B.W.Skin Delivery of Oestradiol from Deformable and Traditional Liposomes:Mechanistic Studies.The Journal of Pharmacy and Pharmacology,51(10) 1123-34.1999; DAYAN, N.and E.TOUITOU.Cariers for Skin Delivery of Trihexyphendyl HCl Ethosomes vs.Biomaterials,21(18):1879–85.2000)。

In addition, sterile fixed oils may be employed as a solvent or suspending medium. For this purpose, any soft fixed oil may be used, including synthetic mono-or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectable compositions and adjuvants, such as local anesthetics, preservatives and/or buffers which may be dissolved in the carrier.

The pharmaceutical forms may also be formulated as suppositories (e.g., for rectal administration). Such compositions may be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ambient temperatures but liquid at the rectal temperature and will therefore dissolve in the rectum to release the drug.

The pharmaceutical form may be formulated to release at a predetermined rate. Immediate release may be obtained, for example, by sublingual administration (i.e., oral administration in a manner such that the active ingredient(s) are rapidly absorbed through the blood vessels of the sublingual plexus).

Controlled release formulations (i.e., formulations such as capsules, pills, or coated tablets, etc., that reduce and/or delay release of the active ingredient(s) after administration) may be administered, for example, orally, rectally, or subcutaneously or by implantation in the target site. In general, controlled release formulations can be obtained by combining the active ingredient(s) with its own matrix material that alters the release rate and/or by using controlled release coatings that delay the breakdown and absorption of the intestinal tract (or the site of the implant), thereby providing a delayed or sustained action over a longer period of time. One such type of controlled release formulation is a sustained release formulation in which at least one active ingredient is continuously released at a constant rate over a period of time. Preferably, the therapeutic agent is released at a rate that maintains the concentration of the therapeutic agent in the blood (e.g., plasma) within a therapeutic range, yet below toxic levels, for a period of at least 4 hours, preferably at least 8 hours, more preferably at least 12 hours. Such formulations can generally be prepared using well known techniques. The carriers used in such formulations are biocompatible and may also be biodegradable. Preferably, the formulation provides a constant level of release of the modulator. The amount of modulator contained in a sustained release formulation depends on, for example, the location of the implant, the intended rate and duration of release, and the nature of the condition to be treated or prevented.

The release rate can be varied using methods well known in the art, including (a) variation in the thickness of the composition of the coating, (b) variation in the amount of plasticizer added to the coating, (c) inclusion of other ingredients, such as agents that modify release, (d) variation in the composition, particle size or particle form of the matrix, and (e) providing more than one passageway through the coating. The amount of modulator contained in a sustained release formulation depends, for example, on the method of administration (e.g., the location of the implant), the intended rate and duration of release, and the nature of the condition to be treated or prevented.

The matrix material, which may or may not itself exert a controlled release function, is generally any material that supports the active ingredient(s). For example, materials such as glyceryl monostearate or glyceryl distearate may be employed. The active ingredient(s) may be combined with the matrix material prior to forming the dosage form (e.g., pill). Alternatively or additionally, the active ingredient(s) may be coated on the surface of microparticles, granules, spheres, microspheres, pellets or pellets comprising a matrix material. Such a coating may be obtained in a conventional manner, for example by dissolving the active ingredient(s) in other or another suitable solvent and spraying. Optionally, additional ingredients are added prior to coating (e.g., to help bind the active ingredient(s) to the matrix material). The matrix may then be coated with a barrier agent prior to applying the controlled release coating. If desired, a plurality of coated matrix units may be encapsulated to produce a final dosage form.

The controlled release coating may be a continuous and uniform film, capable of supporting pigments and other additives, non-toxic, inert and non-adhesive. Coatings that modulate the release of the modifying agent include pH-independent or pH-dependent coatings that can be used to release the modifying agent in the stomach and enteric coatings (allowing the formulation to pass intact through the stomach, in the small intestine, the coating dissolves and the contents are absorbed by the body). The pH-dependent coating includes, for example, shellac, cellulose acetate phthalate, polyvinyl acetate phthalate, methyl hydroxypropyl cellulose phthalate, copolymers of methacrylic acid and esters of zein.

In certain forms, the coating is a hydrophobic material, preferably used in an amount effective to reduce hydration of the gelling agent after administration. Suitable hydrophobic materials include alkyl celluloses (e.g., ethyl cellulose or carboxymethyl cellulose ethers), cellulose ethers, cellulose esters, acrylic polymers (e.g., (poly) acrylic acid, (poly) methacrylic acid, copolymers of acrylic acid and methacrylic acid, copolymers of methyl methacrylate, ethoxyethyl methacrylate, alkanolamide/methacrylic acid copolymers, poly (methyl methacrylate), polyacrylamide, ammonium methacrylate copolymers, aminoalkyl methacrylate copolymers, (poly) methacrylic anhydride and glycidyl methacrylate copolymers), and mixtures thereof.

Aqueous dispersions representative of ethylcellulose include, for example,(FMC Corp., Philadelphia, PA) and(Colorcon, Inc., West Point, PA), both of which are suitable for use in a substrate according to the manufacturer's instructions. Representative acrylic polymers include, for example, various polymers(Rohm America, Piscataway, NJ), either alone or in combination, depending on the release profile desired.

The physical properties of the coating comprising the aqueous dispersion of hydrophobic material may be improved by the addition of more than one plasticizer. Suitable plasticizers for alkyl celluloses include, for example, dibutyl sebacate, diethyl phthalate, triethyl citrate, tributyl citrate, and triacetin. Suitable plasticizers for the acrylic polymer include, for example, citric acid esters, such as triethyl citrate and tributyl citrate, dibutyl phthalate, polyethylene glycol, propylene glycol, diethyl phthalate, castor oil plants, and triacetin.

The controlled release coating is typically applied using conventional techniques, such as by spraying in the form of an aqueous dispersion. If desired, the coating may contain pores or channels to facilitate release of the active ingredient. The pores and channels may be created using well known methods, including the addition of organic or inorganic materials that dissolve, extract or release from the coating in the environment of use. Some of such pore-forming materials include hydrophilic polymers such as hydroxyalkyl celluloses (e.g., hydroxypropyl methylcellulose), cellulose ethers, water-soluble synthetic polymers (e.g., polyvinylpyrrolidone, cross-linked polyvinylpyrrolidone, and polyethylene oxide), water-soluble polydextrose, sugars and polysaccharides, and alkali metal salts.

The amount of active ingredient that can be combined with the material of the carrier to produce a unit dose will vary depending, for example, on the patient to be treated, especially the mode of administration, and any other co-administered drugs. Dosage units typically contain between about 5 μ g and about 2g of the active ingredient. The optimal dosage can be determined using tests and routine procedures well known in the art.

In one aspect of the invention, the composition may contain, in addition to one or more smooth muscle tone-modulating peptides of the invention, one or more other active ingredients including, but not limited to, analgesics, anti-inflammatory agents, anthelmintics, antiarrhythmics, antibiotics, anticoagulants, thrombolytic agents, diuretics, antidepressants, antidiabetics, antiepileptics, antihistamines, antihypertensives, antimuscarinics, antimycotics, antineoplastics, immunosuppressants, immunomodulators, antivirals, anxiolytic sedatives (hypnotics and antipsychotics), beta-adrenoceptor blockers, blood products and substitutes therefor, cardiac inotropic agents (inotropic agents), corticosteroids, antitussives (expectorants and mucolytics), diuretics, dopaminergic agents (antiparkinsonian agents), Hemostatic agents, immunological agents, lipid modulating agents, muscle relaxants, parasympathomimetics, prostaglandins, leukotrienes, bronchodilators, sex hormones (including steroids), antiallergic agents, sympathomimetic agents, vasodilators, prokinetic agents, antiemetics, chemotherapeutic agents, and xanthines.

In some embodiments, the invention includes the use of smooth muscle tone-modulating peptides for the treatment of diseases benefiting from smooth muscle contractility modulation, including but not limited to: erectile Dysfunction (ED), Female Sexual Dysfunction (FSD), Benign Prostatic Hyperplasia (BPH), raynaud's syndrome, Pulmonary Arterial Hypertension (PAH), Systemic Arterial Hypertension (SAH) and hyperreactivity of the airways associated with asthma, COPD, pulmonary fibrosis, silicosis, allergic bronchopulmonary aspergillosis, hereditary angioedema, and neonatal hypoxic respiratory failure.

For example, a method of preparing a pharmaceutical composition, the method comprising introducing any one of the above to a plurality of peptides to a pharmaceutically acceptable excipient in an amount sufficient to treat a condition in which modulation of smooth muscle tone is beneficial.

In some embodiments, the disorder is selected from the group consisting of: erectile Dysfunction (ED), Female Sexual Dysfunction (FSD), Benign Prostatic Hyperplasia (BPH), raynaud's syndrome, Pulmonary Arterial Hypertension (PAH), Systemic Arterial Hypertension (SAH) and hyperreactive asthma with associated airways, COPD, pulmonary fibrosis, silicosis, allergic bronchopulmonary aspergillosis, hereditary angioedema, and neonatal hypoxic respiratory failure.

Use of V-smooth muscle tone-modulating peptides

The smooth muscle tone modulating peptides of the invention are useful in the treatment of diseases that benefit from modulation of smooth muscle tone. In a particular aspect, the peptides of the invention are useful for treating diseases in which smooth muscle tone is permanently or temporarily unbalanced.

In some embodiments, a method for treating a disorder in a patient in need of modulation of smooth muscle tone, comprising administering to the patient a therapeutically effective amount of any one or more of the peptides described above.

The following clinical conditions non-exhaustively illustrate possible therapeutic applications of the peptides in question.

A) ED: relaxation of the cavernous smooth muscle of the penis is a common event in the pharmacological class of mechanisms of action most commonly applied to treat ED. For example, PDE5i (e.g., sildenafil and tadalafil) prevents degradation of cyclic guanosine monophosphate (cGMP) produced by guanylate cyclase following activation by NO and enhances relaxation of the corpus cavernosum of the penis, thus facilitating or enhancing erection. However, PDE5i requires a stimulus capable of inducing the release of NO from the penile nerve and local vascular endothelium. Thus, PDE5i was not effective in patients with nerve or endothelial damage, such as patients with hypertension, diabetes, or patients who received prostatectomy with further nerve damage (SHAMLOUL, R.; GHANEM, H.Erectile dysfunction).The Lancet,381(9861):153-165,2013). The peptides of the invention promote relaxation of smooth muscle tissue even in the presence of nerve or endothelial injury. Thus, the peptides are fully applicable for the treatment of ED, including a phenotype resistant to PDE5 i. Furthermore, the mechanism of action of the smooth muscle tone-modulating peptide favours a synergistic effect with PDE5i, allowing clinical co-administration.

B) And (3) FSD: FSD may be associated with vascular damage in the arterial network that infiltrates the vagina and clitoris, the latter constituting a key structure that promotes orgasm in women. Due to anatomical similarity to the penis, the clitoris is also known to relax in response to stimuli by NO-dependent mechanisms (PARK, k., GOLDSTEIN, i., ANDRY, c., SIROKY, m.b., KRANE, r.j., azazoi, k.m. systemic pathogenic bacteria location for variable ability administration and clinical efficacy).International Journal of Impotence Research,9,27-38,1997). Furthermore, vasodilatation and the consequent increase in local blood flow is beneficial for lubrication and pleasure (VELTEN, J., CHIVERS, M.L., BROTTO, L.A. Does reproduced Testing Impact communication Between Genital and Self-Reported Sexual Arousal in Women?Archives of Sexual Behavior.47(3):1-10.2018). Thus, the peptides of the present invention are usefulTreating FSD.

C) BPH: prostatic hyperplasia is a characteristic of BPH, resulting in increased intra-urethral pressure, impeding urine outflow. Eventually, the urinary tract is completely obstructed, causing an acute urinary retention event as an acute complication of BPH. Treatment of acute urinary retention is also based on mechanisms that can cause relaxation of the smooth muscle of the urethra, thereby reducing the pressure in the urethra (THOMAS, k., CHOW, k., KIRBY, r.s. acid urinary retention: a review of the urinary and management.Prostate cancer and prostatic diseases,7(1):32.2004). Given the activity of the peptides of the invention on smooth muscle, they are expected to be useful in the treatment of BPH.

D) PAH: PAH is a rare, severe, chronic cardiopulmonary disease that is caused by cellular proliferation and fibrosis of the small pulmonary arteries, particularly proliferation and hypertrophy of smooth muscle cells. PAH is clinically recognized by a gradual increase in pulmonary vascular resistance due to structural changes. Current therapeutic options for PAH target mainly three signaling pathways: prostacyclin, endothelin-1, and NO. All of these pharmacological classes modulate vasomotor tone (vasomotor tone), promoting relaxation of blood vessels and reduction of vascular resistance (LAU, e.m.t., giannoultou, e.g., CELERMAJER, d.s., HUMBERT, m.epidemic and treatment of pulmony atrial hypertension).Nature Reviews Cardiology,14(10):603-614.2017). PAH is further divided into five groups by the world health organization according to its etiology. The largest group is group 1 PAHs, which includes idiopathic PAH (ipah), hereditary PAH, drug-and toxin-induced PAH, PAH associated with other diseases, and Persistent Pulmonary Hypertension (PPHN) in newborns. Among them, iPAH has the highest incidence. (CHESTER, A.H., YACOUB, M.H., MONCADA, S.Nitric oxide and pulmony aromatic hypertension.Global Cardiology Science and Practice,14.2017). PAH has been shown to be associated with strong systemic inflammation, with patients exhibiting elevated levels of circulating inflammatory cytokines including IL-1, IL-6, IL-8, TNF-a (PRICE, l.c., WORT, s.j., PERROS, f.,P.,HUERTAS,A.,MONTANI,D.,COHEN-KAMINSKY,S.,HUMBERT,M.Inflammation in pulmonary arterial hypertension.CHEST,141(1):210-221.2012). The peptides of the invention are useful for treating PAH, as they can promote vasodilation of pulmonary arteries, reduce pulmonary artery pressure, ameliorate secondary cardiovascular changes associated with PAH, and reduce inflammation.

E) SAH: the pathological increase in blood pressure leads to a deregulation of the cardiac load (cardiac debt) and/or the resistance of the surrounding vessels. Currently available therapies target one or both components of blood pressure. Different pharmacological classes have been adopted to reduce peripheral resistance, however a large proportion of them promote relaxation of the smooth muscle of the blood vessel or block the effects of contractile stimuli (e.g. those emitted by the sympathetic ANS) (see GOODMAN, l.s. and gimman, a.goodman)&Gilman's pharmacological basis of therapeutics.New York: McGraw-HillP.846,2006). The smooth muscle tension-regulating peptides of the invention are capable of relaxing vascular structures and improving cardiovascular characteristics secondary to hypertension and are therefore useful in the treatment of SAH.

F) Raynaud's syndrome: the physiopathological feature of the disease is ischemic spasms in the limbs of the hands caused by cold or emotional stress. The proposed treatment is based on the reversal of local vasoconstriction by topical application of vasodilators such as nitrates, PDE5i, calcium channel blockers and prostaglandins: (M.,M.Recent achievements in the management of Raynaud’s phenomenon.Vasc Health Risk Manag.6:207-214.2010). Therefore, the peptides of the present invention, which are capable of promoting smooth muscle relaxation and subsequent vasodilation, are useful for the prevention and treatment of Raynaud's disease.

G) Airway hyperreactivity: the expression "hyperresponsiveness" means an increase in airway contractility in the face of sphincter stimulation. This phenomenon is common in various diseases of the respiratory system, where asthma, COPD, bronchitis, and allergies may be notedRhinitis, etc. Hyperreactivity of the airways is mediated by a layer of soft muscular tissue that covers the upper airways and extends to the bronchioles. Treatment involves asymptomatic control of the inflammatory process leading to hyperresponsiveness of the airways, however recovery from crisis with bronchodilators promotes relaxation of adjacent smooth muscles (see BRAMAN, s.s., BARROWS, a.a., DECOTIIS, b.a., SETTIPANE, g.a., CORRAO, w.m. air hyper responsiveness in adaptive inflammation: a risk factor for asthma).Chest,91(5):671-674.1987；POSTMA,D.S.,and KERTJENS,H.A.M.Characteristics of airway hyperresponsiveness in asthma and chronic obstructive pulmonary disease.American journal of respiratory and critical care medicine,158(supplement_2):S187-192,1998；INMAN,M.D.Airway hyperresponsiveness.CHEST Journal123(3) 411S-416 S.2003. The peptides of the invention reverse airway constriction by spasmodic stimulation without eliciting an inflammatory response, and therefore these substances may be used at least for the treatment of symptoms of diseases of the respiratory system with an inflammatory component, examples of which are asthma and COPD.

H) Interstitial lung disease (pulmonary fibrosis): pulmonary fibrosis is one of the subtypes of interstitial lung disease. These diseases are characterized by intense fibroplasia due to lung injury, followed by an inflammatory process, followed by fibroplasia and fibrosis as described above (REYNOLDS, h.y. gail, d.b., KILEY, j.p.Sarcoidosis Vasc.Diffuse Pulm.Dis.,22(1):5-12). It was described in the earlier literature that increased expression of iNOS is noted in the pathogenesis of disease and is considered an important marker of disease progression (2005; HSU Y.C., WANG, L.F., CHIEN, Y.W. Nitric oxide in the pathobiology of diabetes mellitus.Free Radic Biol Med,42(5):599-607,2007). Later on, using mice knockout of these three NOS subtypes and bleomycin treatment (an experimental method for inducing pulmonary fibrosis in animals), it was found that mice knockout of these three NOS subtypes had a worse prognosis than healthy animals, and therefore, the peptide composition of the present invention has a potential to act on potential inflammation and currently unknown factors, and is useful for treating pulmonary fibrosisCandidates (NOGUCHI, s., YATERA, k., WANG, k.y., ODA, k., AKATA, k., YAMASAKI, k., KAWANAMI, t., ISHIMOTO, h., TOYOSHIRA, y., shimowa, h., YANAGIHARA, n., tsutsutsui, m., MUKAE, h.nitrooxide extrits protective effects against blue-induced porous firosis in mice.Respir Res,15(1)；92；2014)。

I) Silicosis: occupational exposure to crystalline silica dust can lead to silicosis, a chronic lung disease. Once the silica particles are inhaled, they cause persistent inflammation of the alveoli and pulmonary fibrosis (THAKUR a.s., BEAMER c.a., MIGLIACCIO c.t., HOLIAN a.critical of MARCO on crystalline silica-induced pulmonary inflammation).Toxicol.Sci462,471; 2009). Silica particles induce macrophage activation, resulting in increased recruitment of neutrophils, lymphocytes, and fibroblasts, which is responsible for fibrosis. Until the present specification, there is no effective therapy for treating pulmonary fibrosis or for altering the progression of the disease (GREENBERG m.i., WAKSMAN J., CURTIS J. (2007).Dis.Mon.(53):394–416；2007；LEUNG,C.C.,YU,I.T.,CHEN,W.Silicosis.Lancet,379(9830):2008-2018；2012；CARNEIRO,P.J.,CLEVELARIO,A.L.,PADILHA,G.A.,SILVA,J.D.,KITOKO,J.Z.,OLSEN,P.C.,CAPELOZZI,V.L.,ROCCO,P.R.,CRUZ,F.F.Bosutinib Therapy Ameliorates Lung Inflammation and Fibrosis in Experimental Silicosis.Front Physiol2017Mar 15; 8:159.2017). The bronchodilator effect of the present invention secondary to smooth muscle cell relaxation in the tracheal ring is useful for symptomatic patients with airflow obstruction without causing an increase in lung tissue inflammation.

J) Allergic bronchopulmonary aspergillosis (ABPA): ABPA is a pulmonary disorder characterized by hypersensitivity to Aspergillus fumigatus (Aspergillus fumigatus) in patients with asthma and cystic fibrosis. It is manifested by uncontrolled asthma and repeated episodes of pulmonary infiltration, leading to wheezing, hemoptysis, productive cough, low fever, weight loss, systemic weakness, and fatigue (AGARWAL, r. chakrabarti, a., SHAH, a., GUPTA, d., MEIS, j.f., GULERIA, r.s., MOSS, r.s., DENNING, d.w., and For the ABPA compatible activity ish ISham working groupis:review of literature and proposal of new diagnostic and classification criteria.Clinical Et Experimental Allergy,(43):850-873,2013). In cystic fibrosis patients who also have a high risk of developing ABPA, exhaled nitric oxide is lower than in low risk patients, indicating that the aflatoxins down-regulate iNOS (LIM, a.y., chamfers, d.c., AYRES, j.g., STABLEFORTH, d.e., honeybee, d.exotic nitrile oxide in cytotoxic fibrosis patients with allogenic branched inhalation allergy aspergillosis.Respir Med.97(4):331-6,2003). Furthermore, the bronchodilator effect of the present invention secondary to smooth muscle cell relaxation in the tracheal ring is useful for patients with airflow obstruction without causing an increase in lung tissue inflammation.

L) neonatal hypoxic respiratory failure: this is a neonatal condition that may benefit from the present invention. Neonatal Persistent Pulmonary Hypertension (PPHN) is the failure of normal pulmonary vascular adaptation at or shortly after birth, resulting in high pulmonary vascular resistance. PPHN treatment is aimed at reducing pulmonary vascular resistance, for which different forms of mechanical respirators have been used. In the case of failure, intravenous vasodilators (tolazoline, epostine alcohol and enoximone) and PDE5i were used. However, these compounds have an adverse risk profile, known to develop hypotension, renal failure, and hemorrhage in certain patients. Inhaled nitric oxide administration is safer for such patients, however, the poor lung expansion of these patients compromises the underlying outcome (muraa, m.c., NEGRO, s., SUN, b., BUONOCORE, g.nitic oxide in neostructural respiratory failure).The Journal of Maternal-Fetal and Neonatal Medicine25(S (1)): 47-50.2012). The peptides described in the present invention may have better safety due to NO systemic exposure when administered intranasally by a suitable device or inhaler, and have the potential to be more effective than inhalation of nitric oxide due to the fact that the peptides of the present invention are able to induce the expression of the NOS isoform, thus producing NO that is locally available.

Thus, the present invention also contemplates the use of smooth muscle tone-modulating peptides to treat individuals suffering from the above-described conditions.

The invention also contemplates the use of a smooth muscle tone-modulating peptide in the manufacture of a medicament for the treatment of the above-mentioned diseases.

The biological activity of smooth muscle tone-modulating peptides, analogs and derivatives thereof can be verified by different methods ex vivo (ex vivo) and in vivo (in vivo). The following examples describe the ex vivo relaxant activity of smooth muscle tone-modulating peptides in an "isolated organ" model based on tracheal rings, pulmonary arteries and penile cavernous bands, fragments rich in smooth muscle tissue structure, without triggering inflammation. The following further examples describe the vasodilation, improvement of hemodynamic parameters and the in vivo effect of pulmonary arterial pressure reduction due to anti-inflammatory effects of selected smooth muscle tone modulating peptides in a rat model of PAH characterized by cardiopulmonary disease caused by elevated pulmonary arterial pressure due to sustained smooth muscle over-contraction.

Accordingly, the invention will be described hereinafter by way of examples, which additionally illustrate the invention, but are not intended to limit the scope of the invention.

Examples

Example 1: synthesis of peptides

The peptide of the invention is synthesized in Rink-amide resin (0.68mmol/g) manufactured by Genone company of Luo, ca.t. Luo, Brazil (batches: P170313-TL569356, P170313-TL569357, P170313-TL569358, P170315-TL569368, P170315-TL569382, P170313-TL569361, P170315-TL569362, P170313-TL569359, P170313-TL569360, P170315-TL569363, P170315-TL569364, P170313-TL569366, P170313-TL 17167, WB170023-P171025, WB170024-P171025, P170315-TL569382, P170315-TL569383, P315-TL 17117117184, P315-TL 80, WB170028 171028, P170024-P17093025, P170170170569381, P170569381, P17056025, P170569381, P170569384, P17056025, P1705632, P170569381, P17056025, P1705632, etc. in solid phase. Final cleavage and deprotection were achieved with water-TFA-1.2-ethanedithiol-triisopropylsilane, 92.5-2.5-2.5-2.5(v/v), 25 ℃ for 180 min. The peptides were extracted with 50% (v/v) acetonitrile in water and purified by Reverse Phase Chromatography (RPC) on a column of Sephasil C8 peptide (5. mu. ST 4.6/100-HPLC) equilibrated with aqueous TFA 0.1%. The sample was eluted using a gradient of acetonitrile with 0.1% de TFA at a flow rate of 2ml/min at 280 nm. After purification, the equilibrium ion exchange of TFA from all compositions can be modified to chloride or acetate ions to maintain all desired physicochemical properties (e.g., solubility, pi, pK, stability, etc.). In addition, all peptides were N-terminally acetylated and C-terminally amidated to increase solubility.

Example 2: in vitro relaxation in airway muscles

The strength of the relaxing effect of peptides was evaluated ex vivo by a spasmodic contraction model of the tracheal ring, which is mainly used to test new bronchodilators (see culum, v.a., far, j.b., JACK, d., LEVY, g.p. salbutimol: a new, selective β -adaptive receptor stimulator stimulin.British journal of pharmacology,35(1):141-151.1969；KAO,C.H.,CHU,Y.H.,WANG,H.W.Effects of lidocaine on rat’s isolated tracheal smooth muscle.European Archives of Oto- Rhino-Laryngology,267(5):817-820.2010；M.A.,I.,ARELLANO-MENDOZA,M.G.,CORREA-BASURTO,J.,TRUJILLO-FERRARA,J.G.Synthesis,pharmacological and in silico evaluation of 1-(4-dihydroxy-3,5-dioxa-4-borabycyclo[4.4.0]deca-7,9,11-trien-9-yl)-2-(tert-butylamine)ethanol,a compound designed to act as aβ2adrenoceptor agonist.European journal of medicinal chemistry,44(7):2840-2846.2009)。

For each experiment, Dunkin-Hartley guinea pigs (400-₂Euthanasia in the atmosphere; the trachea is then exposed, removed and cut into 1 to 3 cartilaginous tracheal ring segments (segments). Each fraction was transferred to a Krebs-containing nutrient solution (NaCl 118 mM; KCl 4.8 nM; CaCl) at 37 ℃ with continuous aeration with a carbonaceous mixture (95% O2 and 5% CO2)₂ 2.5mM；MgSO₄ 1.2mM；KH₂PO₄ 1.2mM；NaHCO₃24 mM; glucose 11mM) in a separate organ bath system. In each system, a bar fixed to the bottom of the vessel and another bar connected to equidistant sensors (GRASS FT-03) constitute the support of the segments (fragments). A data digitizing system connected to equidistant sensors allows recording of the tension variations resulting from tracheal ring contractions (PowerLab)^TM16/30 LabChart, version 8.1, AD Instruments, Australia). Basal tension was adjusted to 1g, and subsequently, the contractility of the fragments was evaluated by stimulation with carbachol (2.5 μ M). After resting for 1 hour, the basal tension was restored, the tissues were subjected to contraction induced by histamine (50 μ M) and then exposed to increasing concentrations of treatment (0.01 to 10 μ M; n ═ 8) or in the comparative PnTx (19).

Table 1 lists the efficacy values (pEC50) expressed as the negative logarithm of the concentration that reduces the voltage to 50% of the maximum histamine-induced contraction, and the mean ± SEM of the efficacy (Emax) calculated from at least 8 independent experiments using the sigmoid nonlinear regression equation of the Software Graph Prism 5.0(Graph pad Software, La Jolla, Ca, USA). The results obtained with the comparison PnTx (19) are shown in Table 1.

Table 1: potency (pEC50) and efficacy values (Emax) were obtained from the concentration-response curve of active peptides in the in vitro relaxation of the smooth muscle of the tracheal ring

The presence of the sequences Ile-Aia-Trp as Xaa6, Xaa7, and Xaa8, respectively, is important for the biological role of the peptide. For example, the sequences SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3 and SEQ ID NO 4 have Ile-Aia-Trp between Xaa5 and Xaa9 and are therefore effective in relaxing smooth muscle cells of airway muscles. However, the sequence SEQ ID NO 7 has Ala instead of Ile as Xaa6 and the sequence SEQ ID NO 8 has Ala instead of Trp as Xaa8, so both are inactivated. Likewise, the sequence SEQ ID NO:24 also has Ile-Aia-Trp as Xaa6, Xaa7, and Xaa8, respectively, and is effective in the relaxation of tracheal ring smooth muscle. However, SEQ ID NO 26 has Tyr instead of Trp at Xaa8 position and is thus inactive.

Another aspect of the biological activity of the peptide is Xaa1, Xaa2, and Xaa15 are either independently absent, or Ala, or a basic natural amino acid (Arg, Lys, or His). For example, compared to the active sequences of the peptides SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3 and SEQ ID NO. 4, the sequence SEQ ID NO. 15 has the acidic amino acid Glu at position Xaa1 instead of the basic amino acid and is therefore inactive. Likewise, the sequence SEQ ID NO 16 has Glu at position Xaa15 instead of a basic amino acid and is therefore also inactive.

Another aspect of the biological activity of the peptide is Xaa4, Xaa5, and Xaa9 are each independently absent or an aromatic amino acid (Phe, Trp, or Tyr). For example, the sequences SEQ ID NO 9, SEQ ID NO 10 and SEQ ID NO 11 have the non-aromatic amino acid Ala at positions Xaa4, Xaa5 and Xaa9, respectively, instead of the aromatic amino acid (Phe, Trp or Tyr), as the active sequence of the peptides SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3 and SEQ ID NO 4, and are thus inactivated.

Another aspect of the biological activity of the peptide is Xaa10 independently being absent or being a basic natural amino acid (Arg, Lys, His). For example, the sequence SEQ ID NO. 12 has the nonpolar amino acid Ala instead of the basic amino acid and is therefore inactive compared to the active sequences of the peptides SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3 and SEQ ID NO. 4.

Another aspect of the biological activity of the peptide is Xaa12 is absent or Ala. For example, SEQ ID NO 21 has Leu instead of Ala at position Xaa12 and is therefore inactive when compared to SEQ ID NO 19.

Another aspect of the biological activity of the peptide is Xaa13 and Xaa14 are each independently an absent or uncharged polar amino acid (Asn, Gin, Ser, or Thr). For example. The sequences SEQ ID NO 13 and SEQ ID NO 14 have the apolar amino acid Ala at positions Xaa13 and Xaa14, respectively, instead of the uncharged polar amino acid as active sequence for the peptides SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3 and SEQ ID NO 4, respectively, and are therefore inactive.

Example 3: in vitro relaxation of the Spongilla band

The peptide of the invention was evaluated for its relaxation potential in the smooth muscle of the cavernosal band ex vivo using tissues isolated from rats, and this model was mainly used to test drug candidates for the treatment of ED (ITALIANO, g.,A.,PAGANO,F.A simplified in vitro preparation of the corpus cavernosum as a tool for investigating erectile pharmacology in the rat.Pharmacological research,30(4):325-334.1994；GEMALMAZ,H.,WALDECK,K.,CHAPMAN,T.N.,TUTTLE,J.B.,STEERS,W.D.,ANDERSSON,KE.In vivo and in vitro investigation of the effects of sildenafil on rat cavernous smooth muscle.The Journal of Urology,165(3):1010-1014.2001)。

male rats of Sprague Dawley (SD) line were sacrificed with a guillotine (guillotine); the penis was surgically removed and placed in a solution containing Krebs-bicarbonate (NaCl 118.1 mM; KCl 4.7 mM; KH)₂PO₄ 1.0mM；MgSO₄ 1.0mM；NaHCO₃ 25.0mM；CaCl₂2.5mM and glucose 11.1 mM). The corpus cavernosum is dissected by removing the glans, urethra, corpus cavernosum and dorsal vein and then separated by cutting the fibrous septa between them. At 370C, fill 95% of 0₂And 5% of C0₂In the case of (2X 7 mm) size of the sponge band, each was placed in a bath containing bicarbonate solution-Krebs (pH 7.4) for isolated organs. The tissue is connected to a tension sensor and changes in tension are continuously recorded. With phenylephrine (10)^-5M) contraction of the sponge band followed by relaxation by electrical stimulation at different frequencies (1 to 32 Hz). To illustrate, the peptides SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:19 and SEQ ID NO:24 (10) were used at different concentrations, with or without L-NAME as a non-specific NOS blocker^-10M；10^-8M；10^-6M) stimulation occurred after 10 min incubation. Alternatively, the peptides are at a single concentration (1 ℃)^-8M), n is replaced by the comparator PnTx (19) at 6. Statistical significance of the percent difference in relaxation between treatment and control was assessed by Two-Way analysis of variance (Two-Way ANOVA), followed by Bonferroni post hoc tests. For comparison between treatment and control based on single frequency stimulation, results were evaluated by One-Way analysis of variance (One-Way ANOVA) followed by Bonferroni post-hoc tests. In all cases, the results were considered statistically different for all values p below 0.05.

As described in the art, PnTx (19) is effective in enhancing smooth muscle relaxation promoted by electrical stimulation of the pre-contracted cavernous band (fig. 2). However, the smooth muscle tone-modulating peptides of the present invention are at as low as 1 ℃^-10The electrical stimulation-induced relaxation of the cavernous bands was enhanced at the concentration of M (fig. 3). Thus, the peptides of the invention are at least 100-fold more effective (1 o) when compared at the optimal stimulation frequency range compared to PnTx (19)^-10M to 1o^-8M) (fig. 4). However, the peptides of the invention did not promote the relaxation of cavernous smooth muscle in the presence of L-NAME as a non-specific NOS blocker, indicating that the therapeutic effect of the compounds described herein is mediated by NO.

Although the present invention has been described in a specific manner, it will be apparent to those skilled in the art that various modifications and variations can be made therein without departing from the scope and spirit of the invention.

Example 4: ex vivo relaxation of pulmonary artery

Ex vivo models of pulmonary artery smooth muscle contraction with phenylephrine are very useful for screening candidate compounds for pulmonary diseases that require vasodilation (ALENCAR, a.k., PEREIRA, s.l., montanopol, t.l., MAIA, r.c.,A.E.,LANDGRAF,S.S.,CARUSO-NEVES,C.,FERRAZ,E.B.,TESCH,R.,NASCIMENTO,J.H.,DE SANT'ANNA,C.M.,FRAGA,C.A.,BARREIRO,E.J.,SUDO,R.T.,ZAPATA-SUDO,G.Beneficial effects of a novel agonist of the adenosine A2A receptor on monocrotaline-induced pulmonary hypertension in rats.Br J Pharmacol,169:953–62.2013)。

since this vasodilation can be achieved by NO producing compounds, the extent of application in relaxing smooth muscle from different tissues was demonstrated in ex vivo assays using the pulmonary artery of male Wistar rats. Rats were anesthetized with midazolam and ketamine (2 mg/kg and 100mg/kg, respectively) by intraperitoneal administration. After a lethal dose of thiopentasodium was infused intraperitoneally (50mg/kg), the pulmonary trunk was carefully excised by sternotomy. The pulmonary artery was carefully dissected and connective tissue removed. The artery was connected to a force sensor, placed in a vertical chamber filled with Krebs solution, oxygenated and maintained at 37 ℃.

After 2 hours of stabilization at 1.5 grams of tension, arteries were exposed to increasing doses of phenylephrine (1 nM-101-JM) to obtain maximal smooth muscle contraction, and then to increasing doses of each of the selected peptides at a concentration of 1 nM-101-JM (n-9). Pulmonary artery produced tension was evaluated at 5 minute intervals for each dose (ALENCAR, a.k., perceira, s.l., montanopol, t.l., MAIA, r.c.,A.E.,LANDGRAF,S.S.,CARUSO-NEVES,C.,FERRAZ,E.B.,TESCH,R.,NASCIMENTO,J.H.,DE SANT'ANNA,C.M.,FRAGA,C.A.,BARREIRO,E.J.,SUDO,R.T.,ZAPATA-SUDO,G.Beneficial effects of a novel agonist of the adenosine A2A receptor on monocrotaline-induced pulmonary hypertension in rats.Br J Pharmacol 169:953–62.2013；ALENCAR,A.K.,PEREIRA,S.L.,DA SILVA,F.E.,MENDES,L.V.,CUNHA VDO,M.,LIMA,L.M.,MONTAGNOLI,T.L.,CARUSO-NEVES,C.,FERRAZ,E.B.,TESCH,R.,NASCIMENTO,J.H.,SANT'ANNA,C.M.,FRAGA,C.A.,BARREIRO,E.J.,SUDO,R.T.,ZAPATA-SUDO,G.N-acylhydrazone derivative ameliorates monocrotaline-induced pulmonary hypertension through the modulation of adenosine AA2R activity.International Journal of Cardiology,173(2):154-62.2014). The assay excludes the absence of phenylephrineNow any constricted arteries.

The results of the tests are summarized in table 2 below. The potency values are shown as the negative logarithm of the concentration that reduces the voltage to 50% of the maximum histamine-induced contraction (pEC50) and are expressed as mean ± SEM. The maximum contraction achieved in a given artery is considered to be 100% of the contraction and the effectiveness (Emax) is expressed as the percentage of relaxation associated with this maximum. The results were calculated from at least 3 independent experiments using sigmoid nonlinear regression equation and statistical analysis of the concentration curves was done by nonlinear fitting the logarithm of the relative agonist response. All the above calculations were performed on the Software Graph Prism 5.0(GraphPad Software, La Jolla, Ca, USA). The tensile analysis was performed on the software LabChart7(AD Instruments, Sydney, Australia). As a cut-off to determine biological significance, we considered pulmonary artery relaxation values above 30% as biologically relevant, considered active, and then calculated Emax values (SUN, c.k., LIN, y.c., YUEN, c.m., CHUA, s., CHANG, l.t., SHEU, j.j., LEE, f.y., FU, m., LEU, s., YIP, h.k.enhanced protection against pulmonary hypertension with latent nitrogen and endovenous promoter cells in rates.International journal of cardiology,162(1):45–58.2012；ALENCAR,A.K.,PEREIRA,S.L.,DA SILVA,F.E.,MENDES,L.V.,CUNHA VDO,M.,LIMA,L.M.,MONTAGNOLI,T.L.,CARUSO-NEVES,C.,FERRAZ,E.B.,TESCH,R.,NASCIMENTO,J.H.,SANT'ANNA,C.M.,FRAGA,C.A.,BARREIRO,E.J.,SUDO,R.T.,ZAPATA-SUDO,G.N-acylhydrazone derivative ameliorates monocrotaline-induced pulmonary hypertension through the modulation of adenosine AA2R activity.International Journal of Cardiology,173(2):154-62.2014)。

Table 2: potency (pEC) was obtained from the concentration-response curve of active peptides in the ex vivo relaxation of the smooth muscle of the pulmonary artery₅₀) Sum effective value (E)_MAX)(n＝9)。

Sequence of	pEC₅₀	Effectiveness (Emax) (%)
			SEQ ID NO:17	7.8±3.0	15％±2.6
SEQ ID NO:18	6.5±9.3	18％±9.12
			SEQ ID NO:19	6.9±6.55	24％±9.71
SEQ ID NO:20	6.1±6.9	6％±7.64
			SEQ ID NO:22	5.8±3.1	8％±2.83
SEQ ID NO:23	6.1±6.2	4％±6.07
			SEQ ID NO:24	4.4±6.4	5％±5.96

All tested peptides had absolute values of pulmonary artery relaxation greater than 30%, so they all had some degree of effectiveness. Due to the high effectiveness value of SEQ ID NO 19, SEQ ID NO 19 was selected for further in vivo experiments.

Example 5: SEQ ID NO 19 induces recovery of hemodynamic parameters in vivo in a monocrotaline-induced Pulmonary Arterial Hypertension (PAH) model.

The Monocrotaline (MCT) model is the most widely used animal model in PAH, the oldest and best described model among the available models in existence. This Model offers the advantage of mimicking several key aspects of human PAH including vascular remodeling, smooth muscle cell proliferation, endothelial dysfunction, inflammatory cytokine upregulation, and right ventricular failure (GOMEZ-ARROYO, j.g., FARKAS, l., alhussiaini, a.a., FARKAS, d., kraskas, d., VOELKEL, n.f., BOGAARD, h.j.the Monocrotaline Model of Pulmonary Hypertension in persivision) after a single intraperitoneal or subcutaneous MCT injection.Am J Physiol Lung Cell Mol Physiol,302(4):L363-9.2012)。

The preferred species currently used for studying MCT-induced PAH is rat, the clinical symptoms of which are anorexia, lassitude, weight loss, and tachypnea within 3-7 days after MCT injection. As lung injury and vascular remodeling progress, the animals develop varying degrees of dyspnea, weakness, diarrhea, and peripheral cyanosis (SCHOENTAL r., HEAD, m.a. pathological changes in rates as a result of respiration of patients with monocrotaline.Br J Cancer,9(1):229-37.1955). One week after MCT injection, endothelial injury, inflammatory infiltration and edema were observed, but Pulmonary Arterial Pressure (PAP) was not elevated. Two weeks later, in the third week after drug administration, PAP elevation leads to Right Ventricular (RV) hypertrophy (WEST, j., HEMMES, a. experimental and translational models of pulmonary hypertension.Compr Physiol,1(2):769-82.2011)。

To evaluate the potential benefit of SEQ ID NO 19 in the treatment of PAH, we used adult healthy male Wistar rats, which were then randomized into two groups: monocrotalineInduced PAH (MCT, n-14), where animals received 60mg/kg of MCT injected intraperitoneally (C2401, Sigma Chemical co., St Louis, MO, USA, lot: WXBC4737V) and 2) controls (CTRL, n-7), where animals received a similar volume of saline solution intraperitoneally. This is day 0 of the study. On the same day, animals were subjected to echocardiographic analysis to establish a baseline prior to random assignment. On day 14, another echocardiogram was performed and the PAH animals were further randomized to receive intranasal Saline (SAL) or SEQ ID NO:19(0.06mg/kg) twice daily for 14 days. On day 28, echocardiography, RV outlet region, Left Ventricle (LV) region, and PAT/PET (pulmonary artery acceleration time (PAT)/pulmonary artery ejection time (PET) ratio) were repeated. In PAH disease, an increase in PAP leads to an increase in RV region due to increased pressure, and a decrease in LV region due to decreased blood flow to the left ventricle. PAT/PET ratio determination can predict mild to moderate PAH, and is considered one of the most predictive non-invasive measures (KOSKENVUO, J.W., MIRSKY, R., ZHANG, Y., ANGELI, F.S., JAHN, S., ALASTALO, T.P., SCHILLER, N.B., BOYLE, A.J., CHATTERJEE, K., DE MARCO, T., YEGHIAZARI ANS, Y.A. complex of an environmental diagnostic to invasive measurement in the evaluation of a pulmonary aromatic permeability in a rat model.Int J Cardiovasc Imaging,26(5):509-18.2010). Hemodynamic parameters were determined by invasive RV intubation, including the systolic pressure of the RV (RVSP and dP/dt ratio (derivative of pressure/derivative of maximum time), which correlates to the pressure in the RV.

Experiments were analyzed using two-way anova, followed by Bonferroni post-hoc testing to determine statistical significance. Also, the standard deviation between subjects is depicted in the graphs of fig. 5 and 6.

When compared to the monocrotaline-induced group treated with vehicle (MCT + vehicle), SEQ ID NO:19 increased the PAT/PET ratio to a value similar to that of the untreated group (control), which corresponds to decreasing PAP to a range of normal values (fig. 5A). The improvement occurred after 14 days of treatment with SEQ ID NO 19. A decrease in PAP resulted in a decrease in RV pressure as evidenced by a decrease in the maximum ratio of RVSP and dP/dt for the group treated with SEQ ID NO:19 compared to the MCT + vector group (FIGS. 5B and 5C, respectively).

The treatment with SEQ ID NO:19 also induced a decrease in RV exit region (fig. 6A) and an increase in LV region (fig. 6B) compared to the MCT + vector group, reaching similar values as the control, which means that SEQ ID NO:19 was able to remodel the heart back to normal.

These results indicate that, in addition to being a vasodilator, SEQ ID NO:19 can be used as a potential treatment for PAH and other lung diseases with or without an inflammatory component as described elsewhere herein.

Example 6: in vivo anti-inflammatory action of SEQ ID NO 19

In the experiment described in example 5, animals were sacrificed for autopsy analysis of inflammatory markers.

As discussed in detail elsewhere herein, all lung diseases, including asthma, COPD, and PAH, have some degree of inflammation, which is part of the disease's physiopathology. Drugs intended to treat these diseases do not trigger inflammation, i.e., pro-inflammatory.

One of the mechanisms of anti-inflammatory action is that smooth muscle cell relaxation reduces endothelial permeability, thereby reducing the migration of inflammatory factors in the blood and reducing the inflammatory process (WALLACE, j.l. nitrile Oxide as a regulator of inflammatory processes.Mem Inst Oswaldo Cruz,100:5-9.2005)。

In one fifth of patients with PAH, mediastinal lymph node enlargement (BERGIN, c.j., PARK, k.j. lymph node enlargement in pulmonary hypertension to respiratory thrombosis) is caused by inflammation-associated edema.J Med Imaging Radiat Oncol,52(1):18-23.2008). The edematous organs weighed more due to the increased fluid content, so we weighed several organs of the animal of example 5 after euthanasia. Treatment with SEQ ID NO 19 was able to reduce edema gain induced by MCT treatment on mediastinal lymph nodes, making it indistinguishable from the CTRL group (FIG. 7A).

Samples of the heart of the monocrotaline study were studied to reveal the potential anti-inflammatory effects of SEQ ID NO 19. During homogenization of all samples, the hearts were placed on ice. Lysis buffer containing 1% protease inhibitor cocktail (Sigma) diluted in Phosphate Buffered Saline (PBS) was added at a rate of 1mL for each sample. 1mM sodium orthovanadate (SIGMA) was also added. The tissue was homogenized and then centrifuged at 10,000g for 10 minutes. Supernatants from each sample were collected into sterile assay tubes to determine total protein and cytokine levels (tumor necrosis factor (TNF) -a, Interferon (INF) -) (, and Interleukin (IL) -113).

The total protein value in the samples was determined using the Bradford assay (BioRad, Hercules, CA, USA) according to the manufacturer's instructions. Briefly, the protocol was performed in microplate at a final volume of 1501-Jl. Calibration curves ranging between 0.025 to 2.0mg/ml were prepared with bovine serum albumin (BSA, SIGMA). Protein solutions were assayed in duplicate. Samples were diluted 20-fold into lysis buffer and 251-Jl from these samples was loaded into each well. A multi-channel pipette is used to add 1251-Jl of Quick StartTM Bradford 1 Xdye reagent solution to produce a final volume of 1501-Jl. The samples were incubated at room temperature for 5 minutes and then the absorbance was measured at 595nm using a spectrophotometer (SpectraMax Microplate reader, Molecular Devices, San Jose, Calif., USA). The standard curve was plotted as a linear regression line and interpolated with the mean absorbance for each sample to determine the total protein concentration of the sample, expressed as mg/ml.

The concentrations of TNF-a, INF-) (, and IL-113 of the heart homogenates were determined using an enzyme-linked immunosorbent assay (ELISA). TNF-a (catalog number 900-K54) and INF-Y (catalog number 900-K98) kits were purchased from Peprotech (Rocky Hill, CO, USA), while IL-113(DY401) kits were purchased from R & D systems (Minneapolis, MI, USA). And measured according to the manufacturer's recommendations.

Data analysis was performed using a statistical Software package (Prism version 5.0, Graph-Pad Software, San Diego, CA). Data are presented as mean ± SEM. All experiments were performed using one-way analysis of variance (ANOVA) followed by Bonferroni multiple comparison test. Statistical differences were considered significant if p < 0.05.

In cardiac homogenates, the MCT group showed an increase in all cytokines assayed (TNF-a, INF-) (, and IL-113) compared to the control group. SEQ ID NO 19 reduced the cytokine to a value similar to the control (FIGS. 7B, 7C, 7D).

Example 7: in vivo anti-inflammatory action of SEQ ID NO 19 and SEQ ID NO 24 compared to PnTX (9)

According to Haspelagh et al (HASPESLAGH, E., DEBEUF, N., HAMMAD, H., LAMBRECHT, B.N. Murine Models of Allergical Ashma.Methods Mol Biol1559:121-136.2017), male a/J mice (18-20 g) were sensitized to House Dust Mites (HDM) to produce acute allergic asthma. Inflammatory cell infiltration in bronchoalveolar lavage fluid (BALF) was analyzed by flow cytometry.

Mice (n-5 per group) were treated with vehicle (PBS), SEQ ID NO:19, SEQ ID NO:24 or PnTx (19) at a dose of 10nMol and 30nMol per animal by intratracheal instillation and material was collected 6 hours after challenge.

Mice exposed to PnTx (19) (30nMol) showed a large increase in total leukocyte number in BALF, mainly due to accumulation of neutrophils (fig. 8). Treatment with SEQ ID NO 19 and SEQ ID NO 24 did not induce accumulation of neutrophils in the bronchoalveolar space 6 hours after intratracheal instillation of the peptides (fig. 8), indicating that these two peptides, unlike the comparator PnTx (19), have NO pro-inflammatory effect in the mouse lung. Notably, chronic inflammation of asthma and other lung diseases such as COPD mainly involves infiltration of neutrophils, the main inflammatory cells involved in this process, into the small airways.

One possible mechanism of anti-inflammatory action is that relaxation of smooth muscle cells can decrease the permeability of endothelial cells, thereby reducing the migration of inflammatory factors in the blood and reducing the inflammatory process. Thus, effective peptides that cause more effective vasodilation than PnT (x)19, such as SEQ ID 24 and SEQ ID:19, have a stronger anti-inflammatory effect.

List of sequences (X)

1 Arg-Aia-Tyr-Phe-Trp-Ile-Aia-Trp-Tyr-Lys-Leu-Aia-Asn-Ser-Lys

2 Arg-Gin-Tyr-Phe-Trp-Ile-Aia-Trp-Tyr-Lys-Leu-Aia-Asn-Ser-Aia

3 Arg-Gin-Aia-Phe-Trp-Ile-Aia-Trp-Tyr-Lys-Leu-Aia-Asn-Ser-Lys

4 Ala-Gin-Tyr-Phe-Trp-Ile-Aia-Trp-Tyr-Lys-Leu-Aia-Asn-Ser-Lys

5 Arg-Gin-Tyr-Phe-Trp-Ile-Aia-Trp-Tyr-Lys-Leu-Aia-Asn-Ser-Lys

6 Arg-Gin-Tyr-Phe-Trp-Ile-Aia-Trp-Tyr-Lys-Ile-Aia-Ser-Asn-Lys

7 Arg-Gin-Tyr-Phe-Trp-Aia-Aia-Trp-Tyr-Lys-Leu-Aia-Asn-Ser-Lys

8 Arg-Gin-Tyr-Phe-Trp-Ile-Aia-Aia-Tyr-Lys-Leu-Aia-Asn-Ser-Lys

9 Arg-Gin-Tyr-Ala-Trp-Ile-Aia-Trp-Tyr-Lys-Leu-Aia-Asn-Ser-Lys

10 Arg-Gin-Tyr-Phe-Aia-Ile-Aia-Trp-Tyr-Lys-Leu-Aia-Asn-Ser-Lys

11 Arg-Gin-Tyr-Phe-Trp-Ile-Aia-Trp-Aia-Lys-Leu-Aia-Asn-Ser-Lys

12 Arg-Gin-Tyr-Phe-Trp-Ile-Aia-Trp-Tyr-Aia-Leu-Aia-Asn-Ser-Lys

13 Arg-Gin-Tyr-Phe-Trp-Ile-Aia-Trp-Tyr-Lys-Leu-Aia-Ala-Ser-Lys

14 Arg-Gin-Tyr-Phe-Trp-Ile-Aia-Trp-Tyr-Lys-Leu-Aia-Asn-Aia-Lys

15 Glu-Gin-Tyr-Phe-Trp-Ile-Aia-Trp-Tyr-Lys-Ile-Aia-Asn-Ser-Lys

16 Arg-Gin-Tyr-Phe-Trp-Ile-Aia-Trp-Tyr-Lys-Ile-Aia-Asn-Ser-Giu

17 Gln-Tyr-Phe-Trp-Ile-Aia-Trp-Tyr-Lys-Leu-Aia-Asn-Ser

18 Tyr-Phe-Trp-Ile-Aia-Trp-Tyr-Lys-Leu-Aia-Asn

19 Phe-Trp-Ile-Aia-Trp-Tyr-Lys-Leu-Aia

20 Arg-Gin-Tyr-Phe-Trp-Ile-Aia-Trp-Tyr

21 Tyr-Trp-Ile-Aia-Trp-Tyr-Lys-Leu-Leu

22 Arg-Gin-Tyr-Phe-Trp-Ile-Aia-Trp

23 Trp-Ile-Aia-Trp-Tyr-Lys-Leu

24 Ile-Aia-Trp-Tyr-Lys

25 Ile-Aia-Trp-Tyr-Giu

26 Ile-Aia-Tyr-Tyr-Lys

Gly-Giu-Arg-Arg-Gin-Tyr-Phe-Trp-Ile-Aia-Trp-Tyr-Lys-Leu-Aia-Asn-Ser-

27 Lys

28 Ile-Aia-Trp-Tyr-Lys-Giy-Giy-Giy-Giy-Giy-Ile-Aia-Trp-Tyr-Lys

29 Ile-Aia-Trp-Tyr-Lys-Arg-Giy-Giy-Giy-Giy-Giy-Arg-Lys-Tyr-Trp-Aia-Ile

30 Ile-Aia-Trp-Tyr-Lys-Giy-Giy-Giy-Giy-Giy-Ile-Aia-Trp-Tyr-Lys-Giy-Giy-Giy-Giy-Giy-Ile-Aia-Trp-Tyr-Lys

Sequence listing

<110> biomedical products development company

C, V, M, F, dora siemens support

<120> synthetic peptides, prodrugs, pharmaceutical compositions and uses

<130> 146-023

<150> 62/694,162

<151> 2018-07-05

<160> 30

<170> PatentIn version 3.5

<210> 1

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<221> conserved region

<222> (6)...(8)

<223> Synthesis of-Ile-Ala-Trp-based PRT

<400> 1

Arg Ala Tyr Phe Trp Ile Ala Trp Tyr Lys Leu Ala Asn Ser Lys

1 5 10 15

<210> 2

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<221> conserved region

<222> (6)...(8)

<223> Synthesis of-Ile-Ala-Trp-based PRT

<400> 2

Arg Gln Tyr Phe Trp Ile Ala Trp Tyr Lys Leu Ala Asn Ser Ala

1 5 10 15

<210> 3

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<221> conserved region

<222> (6)...(8)

<223> Synthesis of-Ile-Ala-Trp-based PRT

<400> 3

Arg Gln Ala Phe Trp Ile Ala Trp Tyr Lys Leu Ala Asn Ser Lys

1 5 10 15

<210> 4

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<221> conserved region

<222> (6)...(8)

<223> Synthesis of-Ile-Ala-Trp-based PRT

<400> 4

Ala Gln Tyr Phe Trp Ile Ala Trp Tyr Lys Leu Ala Asn Ser Lys

1 5 10 15

<210> 5

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<221> conserved region

<222> (6)...(8)

<223> Synthesis of-Ile-Ala-Trp-based PRT

<400> 5

Arg Gln Tyr Phe Trp Ile Ala Trp Tyr Lys Leu Ala Asn Ser Lys

1 5 10 15

<210> 6

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<221> conserved region

<222> (6)...(8)

<223> Synthesis of-Ile-Ala-Trp-based PRT

<400> 6

Arg Gln Tyr Phe Trp Ile Ala Trp Tyr Lys Ile Ala Ser Asn Lys

1 5 10 15

<210> 7

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223>

<400> 7

Arg Gln Tyr Phe Trp Ala Ala Trp Tyr Lys Leu Ala Asn Ser Lys

1 5 10 15

<210> 8

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223>

<400> 8

Arg Gln Tyr Phe Trp Ile Ala Ala Tyr Lys Leu Ala Asn Ser Lys

1 5 10 15

<210> 9

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<221> conserved region

<222> (6)...(8)

<223> Synthesis of-Ile-Ala-Trp-based PRT

<400> 9

Arg Gln Tyr Ala Trp Ile Ala Trp Tyr Lys Leu Ala Asn Ser Lys

1 5 10 15

<210> 10

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<221> conserved region

<222> (6)...(8)

<223> Synthesis of-Ile-Ala-Trp-based PRT

<400> 10

Arg Gln Tyr Phe Ala Ile Ala Trp Tyr Lys Leu Ala Asn Ser Lys

1 5 10 15

<210> 11

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<221> conserved region

<222> (6)...(8)

<223> Synthesis of-Ile-Ala-Trp-based PRT

<400> 11

Arg Gln Tyr Phe Trp Ile Ala Trp Ala Lys Leu Ala Asn Ser Lys

1 5 10 15

<210> 12

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<221> conserved region

<222> (6)...(8)

<223> Synthesis of-Ile-Ala-Trp-based PRT

<400> 12

Arg Gln Tyr Phe Trp Ile Ala Trp Tyr Ala Leu Ala Asn Ser Lys

1 5 10 15

<210> 13

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<221> conserved region

<222> (6)...(8)

<223> Synthesis of-Ile-Ala-Trp-based PRT

<400> 13

Arg Gln Tyr Phe Trp Ile Ala Trp Tyr Lys Leu Ala Ala Ser Lys

1 5 10 15

<210> 14

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<221> conserved region

<222> (6)...(8)

<223> Synthesis of-Ile-Ala-Trp-based PRT

<400> 14

Arg Gln Tyr Phe Trp Ile Ala Trp Tyr Lys Leu Ala Asn Ala Lys

1 5 10 15

<210> 15

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<221> conserved region

<222> (6)...(8)

<223> Synthesis of-Ile-Ala-Trp-based PRT

<400> 15

Glu Gln Tyr Phe Trp Ile Ala Trp Tyr Lys Ile Ala Asn Ser Lys

1 5 10 15

<210> 16

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<221> conserved region

<222> (6)...(8)

<223> Synthesis of-Ile-Ala-Trp-based PRT

<400> 16

Arg Gln Tyr Phe Trp Ile Ala Trp Tyr Lys Ile Ala Asn Ser Glu

1 5 10 15

<210> 17

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<221> conserved region

<222> (5)...(7)

<223> Synthesis of-Ile-Ala-Trp-based PRT

<400> 17

Gln Tyr Phe Trp Ile Ala Trp Tyr Lys Leu Ala Asn Ser

1 5 10

<210> 18

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> conserved region

<222> (4)...(6)

<223> Synthesis of-Ile-Ala-Trp-based PRT

<400> 18

Tyr Phe Trp Ile Ala Trp Tyr Lys Leu Ala Asn

1 5 10

<210> 19

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> conserved region

<222> (3)...(5)

<223> Synthesis of-Ile-Ala-Trp-based PRT

<400> 19

Phe Trp Ile Ala Trp Tyr Lys Leu Ala

1 5

<210> 20

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> conserved region

<222> (6)...(8)

<223> Synthesis of-Ile-Ala-Trp-based PRT

<400> 20

Arg Gln Tyr Phe Trp Ile Ala Trp Tyr

1 5

<210> 21

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> conserved region

<222> (3)...(5)

<223> Synthesis of-Ile-Ala-Trp-based PRT

<400> 21

Tyr Trp Ile Ala Trp Tyr Lys Leu Leu

1 5

<210> 22

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<221> conserved region

<222> (6)...(8)

<223> Synthesis of-Ile-Ala-Trp-based PRT

<400> 22

Arg Gln Tyr Phe Trp Ile Ala Trp

1 5

<210> 23

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> conserved region

<222> (2)...(4)

<223> Synthesis of-Ile-Ala-Trp-based PRT

<400> 23

Trp Ile Ala Trp Tyr Lys Leu

1 5

<210> 24

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<221> conserved region

<222> (1)...(3)

<223> Synthesis of-Ile-Ala-Trp-based PRT

<400> 24

Ile Ala Trp Tyr Lys

1 5

<210> 25

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<221> conserved region

<222> (1)...(3)

<223> Synthesis of-Ile-Ala-Trp-based PRT

<400> 25

Ile Ala Trp Tyr Glu

1 5

<210> 26

<211> 5

<212> PRT

<213> Artificial sequence

<400> 26

Ile Ala Tyr Tyr Lys

1 5

<210> 27

<211> 18

<212> PRT

<213> Artificial sequence

<220>

<221> conserved region

<222> (9)...(11)

<223> Synthesis of-Ile-Ala-Trp-based PRT

<400> 27

Gly Glu Arg Arg Gln Tyr Phe Trp Ile Ala Trp Tyr Lys Leu Ala Asn

1 5 10 15

Ser Lys

<210> 28

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<221> conserved region

<222> (1). > 3 and (11). > 13)

<223> Synthesis of-Ile-Ala-Trp-based PRT

<220>

<221> misc _ feature

<222> (6)...(10)

<223> amino acids optionally linked to a poly-Gly linker

<400> 28

Ile Ala Trp Tyr Lys Gly Gly Gly Gly Gly Ile Ala Trp Tyr Lys

1 5 10 15

<210> 29

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> conserved region

<222> (1)...(3)

<223> Synthesis of-Ile-Ala-Trp-based PRT

<220>

<221> misc _ feature

<222> (7)...(11)

<223> amino acids optionally linked to a poly-Gly linker

<400> 29

Ile Ala Trp Tyr Lys Arg Gly Gly Gly Gly Gly Arg Lys Tyr Trp Ala

1 5 10 15

Ile

<210> 30

<211> 25

<212> PRT

<213> Artificial sequence

<220>

<221> conserved region

<222> (1). (3) and (11). (13) and (21). (23)

<223> Synthesis of-Ile-Ala-Trp-based PRT

<220>

<221> misc _ feature

<222> (6). (10) and (16). (20)

<223> amino acids are optionally linked to a poly-Gly linker

<400> 30

Ile Ala Trp Tyr Lys Gly Gly Gly Gly Gly Ile Ala Trp Tyr Lys Gly

1 5 10 15

Gly Gly Gly Gly Ile Ala Trp Tyr Lys

20 25

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