WO2023246703A1 - 肽抑制剂及其用途 - Google Patents
肽抑制剂及其用途 Download PDFInfo
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- WO2023246703A1 WO2023246703A1 PCT/CN2023/101094 CN2023101094W WO2023246703A1 WO 2023246703 A1 WO2023246703 A1 WO 2023246703A1 CN 2023101094 W CN2023101094 W CN 2023101094W WO 2023246703 A1 WO2023246703 A1 WO 2023246703A1
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- Prior art keywords
- peptide
- pharmaceutically acceptable
- solvate
- prodrug
- acceptable salt
- Prior art date
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/10—Peptides having 12 to 20 amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
Definitions
- TGF ⁇ transforming growth factor ⁇
- TGF- ⁇ and its signaling pathway have been proposed as drug therapeutic targets for fibrotic disorders as well as tumors.
- TGF- ⁇ is also involved in a variety of normal physiological processes, which results in high toxicity of drugs that directly target TGF- ⁇ and its signaling pathway.
- drugs that directly target TGF- ⁇ and its signaling pathway.
- some such drug development projects have progressed slowly or even been terminated due to serious drug-related adverse reactions after entering the clinical stage.
- TGF- ⁇ When TGF- ⁇ is synthesized and secreted, it is an inactive precursor structure. It must undergo a series of activation events to become an active fragment before it can bind to its receptor to mediate downstream pathway transduction.
- the activation event of TGF- ⁇ is completed by different protein molecules under different environments and conditions. Therefore, targeting specific proteins involved in activation in disease situations has become the latest research direction to solve the difficulty of making TGF- ⁇ drugs.
- Thrombospondin-1 is a protein that has been clinically found to be actively expressed in fibrosis and tumor-related indications.
- TSP-1 can participate in TGF- ⁇ activation events in chronic inflammation and fibrosis, and is the main activator of TGF- ⁇ 1 in the body (Cell, Vol. 93, 1159–1170, June 26, 1998, Copyright 1998 by Cell Press).
- peptide drugs targeting TGF- ⁇ activation as they act at the site where TGF- ⁇ 1 is activated, are readily catabolized, and have low antigenicity, may provide better therapeutics than other anti-TGF- ⁇ 1 agents.
- TGF- ⁇ 1 activation inhibitory peptide the inhibitory peptide of the present invention
- R 1 is acetyl or absent
- X2 is selected from natural amino acids or unnatural amino acids, preferably, selected from polar side chain amino acids, such as uncharged polar side chain amino acids S, C, G, N, Q, T, Y or negatively charged Polar side chain amino acids D and E are more preferably selected from amino acids N, C, Q, S, T, E and D; more preferably are selected from amino acids Q, C, N, E or S;
- X 5 is E
- X 6 is D
- X 7 does not exist, or is composed of 1, 2, 3, 4, 5, 6 or 7 amino acids selected from the amino terminus to the carboxyl terminus in the amino acid sequence Z 1 Z 2 Z 3 SZ 4 LQ sequence consisting of
- Z 1 is the amino acid N, Q, P, A, L, V, M, or I, preferably N, P or L, most preferably N or P;
- Z3 is amino acid A, V, L, or I, preferably A or V,
- Z 4 is amino acid F, Y, H, or W, preferably F or H;
- 0-1 small side chain amino acids are inserted between X 3 and X 4 , such as P or G, preferably, P is inserted;
- the peptide has 0-2 additional amino acid residues added after X7 , for example, amino acid residues selected from Q, E, D and N 1-2 amino acid residues of the base are added, or 1 amino acid residue selected from C or K is added.
- X 1 does not exist, or is selected from T and S;
- X 2 is selected from amino acids Q, N, C, S, T, E and D;
- X 3 is D
- X 4 is selected from A, C, G, P, S, T and V;
- X 5 is E
- X 6 is D
- X 7 does not exist, or is composed of 1, 2, 3, 4, 5, 6 or 7 amino acids selected from the amino terminus to the carboxyl terminus in the amino acid sequence
- Z 1 Z 2 Z 3 SZ 4 LQ consists of a sequence, where,
- Z 2 is T or C or K,
- Z 3 is A or V
- Z 4 is F, Y, H, or W.
- X 1 is selected from T and S;
- X 2 is selected from Q and N;
- X 4 is selected from A and P;
- X 5 is E
- Z 2 is T, K or C
- Z 3 is A or V, preferably A
- the peptides of the present invention may be linear peptides or cyclic peptides.
- the present invention also provides that the inhibitory peptide of the present invention or a pharmaceutically acceptable salt, solvate or prodrug thereof is used in diseases caused by or related to TGF- ⁇ 1 activation involving TSP-1. (i.e., "TGF- ⁇ 1-related diseases").
- TGF- ⁇ 1-related diseases i.e., "TGF- ⁇ 1-related diseases”
- the inhibitory peptide of the present invention or its pharmaceutically acceptable salt, solvate or prodrug can be used to prepare a medicament for preventing or treating fibrotic disorders.
- the fibrotic disease is characterized by activation of TGF- ⁇ 1 involving TSP-1 and exhibits excessive deposition of extracellular matrix (Extracellular Matrix, ECM), but is not limited to the site where the disease occurs, nor is it limited to traditional disease classification.
- ECM Extracellular Matrix
- the present invention also provides that the inhibitory peptide of the present invention can be used to prepare drugs for preventing or treating cancer related to TGF- ⁇ 1 activ
- Figure 1 shows the HE-stained pathological sections of the fibrosis mouse disease model. The severity of inflammation was evaluated by HE staining, which showed the inhibitory effect of the polypeptide of the present invention on inflammatory infiltration in pathological parts of fibrosis.
- Figure 2 shows the MASSON’S stained pathological sections of the fibrosis mouse disease model. The severity of fibrosis was evaluated through MASSON’S trichrome staining, which showed the inhibitory effect of the polypeptide of the present invention on fibrosis deposition in pathological parts of fibrosis.
- Figure 3 shows the MASSON’S stained pathological sections of the fibrosis rat disease model, showing the inhibitory effect of the polypeptide of the present invention on fibrosis deposition in the pathological parts of fibrosis.
- the term “comprises” or “includes” means the inclusion of the stated element, integer or step, but not the exclusion of any other element, integer or step.
- the term “comprises” or “includes” is used herein, it also encompasses a combination of the stated elements, integers, or steps unless otherwise indicated.
- reference is made to “comprising” a particular sequence, peptides or polypeptides consisting of that particular sequence are also intended to be encompassed.
- peptide and “polypeptide” are used interchangeably and refer to a sequence of amino acids between 2 and 100 amino acids in length that are linked by peptide bonds. Amino acids can be naturally occurring and non-naturally occurring.
- conservative amino acid substitution or “conservative amino acid substitution” means an amino acid substitution that does not adversely affect or alter the biological function of the polypeptide comprising the amino acid sequence.
- a typical conservative amino acid substitution involves replacing one amino acid with another that has similar chemical properties (such as charge or hydrophobicity).
- Conservative substitution tables for functionally similar amino acids are well known in the art.
- conservative substitution residues can be from the following conservative substitution table, especially the preferred conservative amino acid substitution residues in the table.
- the inventors surprisingly found that short peptides with negatively charged amino acid motifs (DXED) can effectively elicit inhibitory effects on TSP-1-dependent TGF- ⁇ 1 activation.
- DXED negatively charged amino acid motifs
- the inventors further identified the essential amino acids and possible substitution sites in the peptide sequence; and through peptide cyclization and/or main chain modification, the structure was optimized and a series of Novel TGF- ⁇ 1 activation inhibitory peptides with improved biological activity and/or physicochemical properties.
- the present invention provides a TGF- ⁇ 1 activation-inhibiting peptide or a pharmaceutically acceptable salt, solvate or prodrug thereof.
- the peptides of the invention contain a negatively charged core motif (DXED) and are 5-13 amino acids long. Preferably, the peptides of the invention consist of 7-11 amino acids.
- the peptides of the invention are linear or cyclic pentapeptides or hexapeptides, such as linear or first cyclized hexapeptides.
- the peptides of the invention are linear or cyclized heptapeptides, such as head-to-tail cyclized heptapeptides.
- the peptides of the invention comprise a proline substitution, preferably at the second residue position of the core sequence DXED; or at the first residue position immediately C-terminal to the core sequence.
- the overall net charge of the peptides of the invention is negative, for example, -1, -2, -3 or -4, preferably -2 or -3.
- the peptides of the invention are cyclic peptides.
- peptides of the invention can be cyclized by covalent linkage of head and tail amino acids.
- the peptides of the invention can be cyclized via a lactam or disulfide bond formed between two amino acid side chains.
- appropriate amino acids or analogues with appropriate side chain groups can be placed at appropriate positions in the peptide of the invention to facilitate intramolecular amide or disulfide bond formation in the peptide.
- the side chain amino group of lysine (K) at the C-terminus of the core motif forms a lactam bond, and the two amino acids forming a ring are separated by no less than 4 amino acids, for example, 4, 5 or 6 amino acids apart.
- the cyclizing lysine residue is the last residue at the C-terminus of the peptide.
- the peptides of the present invention may contain chemical modifications, such as N-terminal acetylation, C-terminal amidation, PEG modification, lipid modification, D-amino acid substitution, or unnatural amino acid substitution.
- peptides of the invention are modified by covalent attachment to molecules that allow the peptide to retain its ability to inhibit TGF- ⁇ 1 activation, including, for example, glycosylation, acetylation, PEGylation, phosphorylation, amidation, or Derivatization using known protecting/blocking groups.
- the modification is N-terminal acylation (especially acetylation).
- the modification is C-terminal amidation.
- the TGF- ⁇ 1 activation inhibitory peptide according to the present invention can inhibit TSP-1-dependent TGF- ⁇ 1 activation, and preferably has at least one of the following properties:
- the peptides of the invention block the binding of TSP-1 to its receptor in an assay as in Example 2.
- the peptides of the invention exhibit a blocking rate of greater than 15%, 20%, 25%, 30%, 35%, 40%, or higher in an assay as in Example 2.
- the peptides of the invention comprise an amino acid sequence corresponding to or consisting of one of SEQ ID NOs: 3-6, 14, 17, 22, 24-26, 31, 33, 36, 40-47. composition. In some embodiments, the peptides of the invention comprise or consist of one of SEQ ID NOs: 3-6, 14, 17, 22, 24-26, 31, 33, 36, 40-47. In some embodiments, the peptides of the invention comprise or consist of an amino acid sequence corresponding to one of SEQ ID NOs: 3, 6, 14, 22, 24-26, 33, 41-46. In some embodiments, the peptides of the invention comprise or consist of one of SEQ ID NOs: 3, 6, 14, 22, 24-26, 33, 41-46.
- the peptides of the invention comprise or consist of an amino acid sequence corresponding to one of SEQ ID NOs: 6, 22, 25-26, 33, 40, 43, 44, 46. In some embodiments, the peptides of the invention comprise or consist of one of SEQ ID NOs: 6, 22, 25-26, 33, 40, 43, 44, 46. In some embodiments, the peptides of the invention comprise or consist of SEQ ID NO: 6. In some embodiments, the peptides of the invention comprise or consist of SEQ ID NO: 22. In some embodiments, the peptides of the invention comprise or consist of SEQ ID NO: 25. In some embodiments, the peptides of the invention comprise or consist of SEQ ID NO: 26. In some embodiments, peptides of the invention comprise or consist of SEQ ID NO: 33. In some embodiments, the peptides of the invention comprise or consist of SEQ ID NO: 43.
- TGF- ⁇ 1 activation inhibitory peptide of the present invention The following are some embodiments of the TGF- ⁇ 1 activation inhibitory peptide of the present invention.
- X 1 does not exist, or is a natural or unnatural amino acid residue
- X 2 and X 4 are each independently a natural or unnatural amino acid residue
- X 3 is D
- X 5 is E
- X 6 is D
- X 7 is a sequence of 0-7 amino acids
- R 2 is amino or absent.
- X 1 does not exist, or is selected from homoserine, allelothreonine, T and S;
- X 3 is D
- X 4 is selected from small side chain amino acids, such as A, C, G, P, S, T and V, more preferably from amino acids A, P and S, more preferably from amino acids A and P;
- X 5 is E
- X 6 is D
- X 7 does not exist, or is composed of 1, 2, 3, 4, 5, 6 or 7 amino acids selected from the amino terminus to the carboxyl terminus in the amino acid sequence Z 1 Z 2 Z 3 SZ 4 LQ sequence consisting of
- Z 1 is the amino acid N, Q, P, A, L, V, M, or I, preferably N, P or L, most preferably N or P;
- Z3 is amino acid A, V, L, or I, preferably A or V,
- Z 4 is amino acid F, Y, H, or W, preferably F or H;
- 0-1 small side chain amino acids are inserted between X 3 and X 4 , such as P or G, preferably, P is inserted;
- the peptide has 0-2 additional amino acid residues added after X7 , for example, independently selected from Q, E, D and N 1 or 2 amino acid residues are added, or 1 amino acid residue selected from C or K is added.
- X2 is selected from the amino acids N, C, homocysteine, Q, S, homoserine, T , allelothreonine, E and D.
- X2 is selected from the amino acid Q, C, N, E or S.
- X4 is a small side chain amino acid, such as A, C, G, P, S, T and V.
- X 7 is an amino acid sequence selected from the group consisting of: N; P; L; NTA; NTV; PTA; NTAS ;PTAS;NTVSF;NTASF;NTASH;NTVSFLQ.
- R 1 is acetyl or absent
- R 2 is amino or absent
- X 1 is T or S
- X 2 is selected from polar side chain amino acids
- X 7 is a sequence consisting of 1, 2, 3, 4 or 5 amino acids selected from the amino acid sequence Z 1 TZ 3 SZ 4 from the amino terminus to the carboxyl terminus, where,
- Z 1 is N, P or L
- Z 3 is A, V, L, or I,
- R 1 is acetyl or absent
- R 2 is amino or absent
- X 1 is T or S
- X 4 is selected from small side chain amino acids, such as A, C, G, P, S, T and V, more preferably from amino acids A, P and S, more preferably from amino acids A and P;
- Z 1 is a natural amino acid or an unnatural amino acid
- X 8 does not exist, or is a sequence of 1-5 amino acids.
- peptide according to embodiments 55-61 or a pharmaceutically acceptable salt, solvate or prodrug thereof wherein A sequence consisting of 2, 3, 4 or 5 amino acids, wherein Z 3 is A, V, L, or I, and Z 4 is F, Y, H, or W, preferably F or H.
- X is D
- Z is K
- the peptide is cyclized through a lactam bond formed between the side chains of X and Z ;
- X7 consists of Z1Z2
- X1 is T or S.
- the invention provides methods for producing inhibitory peptides of the invention. Chemical synthesis, fermentation, or genetic recombination techniques can be used to produce the peptides of the present invention.
- SPPS solid-phase peptide synthesis
- An automatic peptide synthesizer can also be used for synthesis. Compared with recombinantly synthesized peptides, subsequent purification of SPPS peptide products is simpler and easier. Therefore, it is preferred to apply SPPS technology to synthesize the short peptide of the present invention.
- HMBA 4-toluenehydramine
- CTC 2-chlorotritylchloride
- Merrifield resin a variety of functional resins have also been developed to allow peptide cyclization in the solid phase by coupling the resin with different linkers.
- the present invention provides a method for preparing the peptide of the present invention, the method comprising: (1) synthesizing a linear peptide with a prescribed sequence, for example, starting from 4-toluenehydramine (HMBA) resin
- HMBA 4-toluenehydramine
- the starting materials are Fmoc protected amino acids as monomers, hexahydropyridine/DMF solution as deprotection reagent, and under the action of condensing agent and alkaline conditions, amino acids are connected one by one to produce the linear peptide;
- (2) can Optionally, cyclize the linear peptide obtained in step (1); (3) purify the peptide product obtained in step (1) or (2).
- the invention provides a method for preparing the peptide of the invention, the method comprising: (1) recombinantly expressing a linear peptide with a specified sequence; (2) optionally, performing step (1) Cyclization of the linear peptide obtained in step (3) Purification of the peptide product obtained in step (1) or (2).
- the invention also provides nucleic acids comprising amino acid sequences encoding peptides of the invention, vectors (eg, expression vectors) and host cells comprising the nucleic acids.
- Peptide cyclization is a common peptide modification technique that can include various strategies such as head-to-tail cyclization, side chain to side chain cyclization, and backbone to side chain cyclization. Often, a single linear peptide is too flexible without being linked to other peptides. Cyclization organizes intramolecular interactions It can promote the formation of the secondary structure of the peptide, thereby improving the stability of the peptide.
- the peptides of the invention form monocyclic peptides by head-to-tail cyclization.
- the peptide of the invention is formed by side chain cyclization between the side chains of glutamic acid (E) or aspartic acid (D) and lysine (K) Lactam bridge.
- the peptides of the invention form a disulfide bond between the side chains of two cysteine (C) residues via side chain cyclization.
- the lysine amino acid residue is located at the C-terminus of the core sequence of the invention (DXED) and is identical to the first aspartic acid D of the core sequence (DXED).
- the residue forms the lactam bridge, and more preferably, the K residue and the D residue are separated by at least 4 residues, for example, 4, 5, or 6 residues, preferably the lysine residue
- the acid residue is the last residue at the C-terminus of the peptide of the invention.
- the two cysteine residues used for cyclization are located at the N-terminus and C-terminus of the core sequence (DXED), more preferably, the two Cysteine residues are separated by at least 5 residues, for example, 5, 6, 7, 8 residues.
- the amino acid at position 3 of the peptide of the present invention is D and the amino acid at position 8 is K, and the peptide of the present invention forms a lactam bond through the side chains of the two to form a ring.
- peptides After peptides are synthesized, they can be modified with or without the use of medicinal chemistry techniques. In some cases, it may be advantageous to modify the peptide, for example, by modifying to simulate, stabilize, or build a more suitable secondary structure to improve the biological activity of the peptide drug, and/or to improve the selectivity and stability of the peptide drug. properties and solubility.
- the peptide modification may be amino acid substitution or residue modification of non-essential amino acids, and/or modification of the N-terminus and/or C-terminus of the peptide, such as N-terminal acetylation modification and C-terminal amidation modification. Modifications can also be substitutions of key amino acid residues that affect the biological activity of the peptide to seek changes in activity.
- peptides may contain unnatural amino acids.
- the peptide may contain 1, 2, 3, 4, 5, or 6, or more unnatural amino acids, as desired.
- the amino acids making up the peptide can each be independently selected from natural amino acids.
- Chemically synthesized peptides often carry free amino groups and free carboxyl groups.
- the ends of the peptide can be blocked, that is, N-terminal acetylation and C-terminal amidation, to make the synthetic peptide closer to a mimic of the natural protein and improve the stability of the peptide.
- the peptides of the invention may contain D-amino acids, unnatural amino acids, amino acid analogs, and/or group substitutions and modifications; or may be linked to polymers or to pharmaceutical carriers.
- the invention also provides pharmaceutical compositions comprising a peptide of the invention, or a pharmaceutically acceptable salt, solvate or prodrug thereof.
- pharmaceutical compositions may also optionally contain suitable pharmaceutical excipients, pharmaceutical carriers, pharmaceutical excipients, including buffers.
- Routes of administration for peptide administration include, but are not limited to, subcutaneous, intramuscular and intravenous administration, mucosal administration (e.g., nasal administration, pulmonary mucosal administration, sublingual administration), oral administration (e.g., Add gastrointestinal penetration enhancer or carrier), and transdermal administration.
- the peptide can be formulated into any form suitable for administration, such as injection, eg intravenous injection or intravenous drip, lyophilized powder, etc. Excipients for use in different dosage forms are known in the art.
- compositions may also contain other therapeutic agents that are beneficial for the particular disease to be treated.
- the invention therefore also provides pharmaceutical compositions comprising a peptide of the invention, or a pharmaceutically acceptable salt, solvate or prodrug thereof, and further comprising other therapeutic agents beneficial for the particular disease to be treated.
- the peptides of the invention may be combined with such other therapeutic agents. be contained in the same or different compositions; and may be administered simultaneously, sequentially, or in any order and with any dosing regimen with the other therapeutic agents.
- included in the pharmaceutical composition of the present invention may be, in particular, the peptide of the present invention of the aforementioned embodiments 1-91 or a pharmaceutically acceptable salt thereof, especially the peptide of the present invention of one of the aforementioned embodiments 76-86 or A pharmaceutically acceptable salt thereof, in particular, the peptide of the present invention according to one of the aforementioned embodiments 78-86 or a pharmaceutically acceptable salt thereof.
- the term "subject” or “patient” or “individual” includes any human or non-human animal.
- non-human animals includes all vertebrate animals, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cattle, chickens, amphibians, reptiles, and the like.
- the subject of the invention is human.
- treating a disease or condition means ameliorating the disease or condition (ie, slowing or arresting or reducing at least one of the progression of the disease or its clinical symptoms). In other embodiments, “treating” refers to alleviating or improving at least one physical parameter, including those physiological parameters that may not be discernible by the patient. In other embodiments, “treating” or “treating” refers to modulating a disease or disorder physically (eg, stabilization of discernible symptoms), physiologically (eg, stabilization of body parameters), or both. Unless explicitly described herein, methods for assessing treatment and/or prevention of disease are generally known in the art.
- prevention of a disease or disorder includes inhibition of the onset or progression of the disease or disorder or symptoms of a particular disease or disorder.
- prevention refers to the administration of a drug before the onset of signs or symptoms of fibrosis or cancer, especially in subjects at risk of developing the disease.
- the invention provides methods for preventing or treating TGF- ⁇ related diseases, comprising administering to a subject in need thereof an effective amount of a peptide according to the invention or a pharmaceutically acceptable salt or solvate thereof or prodrugs or pharmaceutical compositions according to the invention.
- the method further comprises administering to the subject an effective amount of a second therapeutic agent, such as a chemotherapeutic agent or an immunotherapeutic agent.
- a second therapeutic agent such as a chemotherapeutic agent or an immunotherapeutic agent.
- the TGF- ⁇ -related disease treated according to the method of the present invention has macrophage infiltration in the lesion site.
- the TGF- Beta-related diseases are associated with tissue damage, inflammation, or fibrosis.
- the TGF- ⁇ -related disease involves metastasis of tumor cells.
- the condition associated with pathological increase in TGF- ⁇ activity is fibrosis.
- the invention provides a peptide of the invention or a pharmaceutically acceptable salt, solvate or prodrug thereof or a pharmaceutical composition of the invention for use as a medicament for preventing or treating a fibrotic disorder in a subject, and corresponding treatment and prevention methods.
- the known diseases with fibrosis characteristics involve a very large spectrum of diseases, which can be classified according to the tissues and organs in which they occur, such as fibrosis of lungs, liver, kidneys, bone marrow and other tissues; they can also be classified as common Disease classification, such as chronic inflammatory diseases, tumor diseases, immune diseases, etc.
- the pathological characteristic of fibrosis is being used as an important basis for reclassifying or even naming, diagnosing, and treating fibrotic diseases, and it is no longer limited by the recognition of the location of the disease, or limited to traditional disease classification.
- a "fibrotic disorder” refers to a disease characterized by a fibrotic pathological phenotype and is not limited to the specific site of disease occurrence or traditional classification of the disease.
- fibrotic diseases that can be treated by the method of the present invention include, but are not limited to, fibrosis of various tissues and organs, such as pulmonary fibrosis, liver fibrosis, and renal fibrosis; as well as chronic inflammation, tumors, and immune diseases.
- the invention provides methods for preventing or treating a fibrotic disorder in a subject, comprising administering to a subject in need thereof a prophylactically or therapeutically effective amount of a peptide according to the invention or a pharmaceutical thereof. acceptable salts, solvates or prodrugs or pharmaceutical compositions according to the invention.
- the fibrotic disorder is liver fibrosis, pulmonary fibrosis, renal fibrosis, myelofibrosis, skin fibrosis, or cardiac fibrosis.
- the fibrotic disorder is pulmonary fibrosis, such as idiopathic pulmonary fibrosis (IPF).
- the method includes administering the peptide of the invention to the subject by inhalation, for example, as a nebulized formulation.
- the cancer is sarcoma, pancreatic cancer, glioblastoma, head and neck cancer, melanoma, breast cancer, or colorectal cancer.
- the cancer is selected from squamous cell carcinoma, epidermoid carcinoma, urothelial carcinoma, adenocarcinoma, adrenocortical carcinoma, basal cell carcinoma, ductal carcinoma in situ (DCIS), invasive ductal carcinoma, thymic carcinoma, and Renal cell carcinoma.
- the synthesized linear and cyclic crude peptides were separated and purified by HPLC, in which C18 column was selected, eluent A of 0.1% TFA/acetonitrile solution, and eluent B of 0.1% TFA/water solution. The main peaks are collected, and the molecular weight of the product is detected by mass spectrometry to match the theoretical value. After freeze-drying, the high-quality peptide is obtained.
- the synthesized polypeptide sequence is shown in Table 1, in which the peptide represented by each sequence number is composed of the "sequence" part and the "modification" part in the sequence description.
- the ELISA method was used to detect the ability of the polypeptide to block the binding of TSP-1 protein and TSP-1 receptor protein CD36.
- FITC-labeled anti-Fc tag antibody Beyotime, Cat. No. A0556
- TECAN model Infinite 200Pro
- the blocking rate of the peptide blocking the binding of CD36 to TSP-1 protein was obtained, that is, (fluorescence signal of the control group - fluorescence signal of the peptide-treated group)/control group
- the fluorescence signal ⁇ 100% was used to evaluate the ability of the polypeptide to block the binding of CD36 to TSP-1 protein (Table 2-1).
- This example uses a cell model with high TSP-1 expression to detect the inhibitory effect of polypeptides on TSP-1-dependent TGF- ⁇ activation.
- the experiment used Phorbol 12-myristate 13-acetate (PMA) (Sigma, Cat. No. P1585) to induce the differentiation of THP-1 cells (American Type Culture Collection, ATCC, TIB-202) into cells with obvious macrophage expression. Characteristic macrophage-like cells.
- PMA Phorbol 12-myristate 13-acetate
- THP-1 cells American Type Culture Collection, ATCC, TIB-202
- Characteristic macrophage-like cells Characteristic macrophage-like cells.
- TSP-1 protein expression increased significantly and L-TGF- ⁇ was secreted; in the absence of factors that interfere with L-TGF- ⁇ activation, the plasmin secreted by the cells will recognize and cleave the CD36 receptor on the cell surface. body-bound TSP-1/L-TGF- ⁇ complex, thereby producing increased amounts of active TGF- ⁇
- Example 4 Inhibition test of active TGF ⁇ content by polypeptides in fibrosis mouse disease model
- This example is to evaluate the inhibitory effect of polypeptides on the content of active TGF- ⁇ during the progression of fibrotic diseases.
- This example uses a mouse pulmonary fibrosis model induced by bleomycin (BLM).
- BLM mouse pulmonary fibrosis model induced by bleomycin
- the BLM animal model has the advantages of high similarity with the pathological characteristics of the primary disease, easy operation, and good reproducibility. It is the most representative fibrosis mechanism model and is also a recognized and recommended animal model for drug efficacy research.
- the administration procedure was as follows (An Official American Thoracic Society Workshop Report: Use of Animal Models for the Preclinical Assessment of Potential Therapies for Pulmonary Fibrosis, Am J Respir Cell Mol Biol Vol 56, Iss 5, pp 667–679, May 2017): Used A small animal anesthesia machine (Reward, model R500IE) atomized isoflurane (Reward, batch number 22041701) and then anesthetized the experimental animals. Take an animal in a suitable anesthetized state and place the animal's head upward and its body tilted on its back, and hang it on the operating platform through its incisors (the operating platform is tilted at about 60°).
- the operator holds tissue forceps in his right hand and gently picks up the tongue and pushes it outward and upward to open the animal's mandible. He holds a laryngoscope (Yuyan, model SR310-RW) in his left hand and slides the leaf-shaped front blade of the laryngoscope into the mouth along the center of the tongue body to the vocal cords. Gently press the tongue body and tongue base in front of the door and upward, insert the needle of the applicator from the mouth against the laryngoscope into the glottis of the animal, and quickly inject 50 ⁇ L of the liquid in the applicator.
- a laryngoscope Yamayan, model SR310-RW
- EK0515 EK0515 to measure the active TGF- ⁇ content in the lung tissue of mice in each group, and calculate the ratio of active TGF- ⁇ content in the peptide administration group and the model group. , to evaluate the effect of peptides on active TGF- ⁇ content.
- Table 4 In the fibrosis disease model, the content of active TGF- ⁇ in the polypeptide administration group was reduced to varying degrees compared with the model group, indicating that the polypeptide of the present invention has an effect on active TGF- ⁇ induced by lung injury during the progression of fibrosis. Increased beta content has an inhibitory effect.
- TGF- ⁇ intracellular signal transduction depends on the phosphorylation events of Smad family proteins, among which the increase in Smad2/3 phosphorylation content is an important indicator of the occurrence of TGF- ⁇ signaling pathway transduction.
- Example 6 Inhibitory test of collagen deposition by polypeptides in fibrosis mouse disease model
- This example evaluates the inhibitory effect of polypeptides on collagen deposition in fibrotic tissue.
- 6-8 weeks old 18-20g male C57/6J mice were used as the research subjects.
- the experimental animals were randomly divided into groups, with 8 animals in each group.
- On the day of modeling (Day 1), a single dose of 0.7 USP/kg bleomycin solution was given to the trachea to create the model; on Day 8, treatment and administration began, and each peptide administration treatment group was given a 3 mg/kg dose of peptide drug in the trachea, with a dosing frequency of every week. 2 times, a total of 4 times of administration.
- the BLM model group was given an equal volume of solvent (physiological saline) as a control.
- the operation method was the same as described in Example 4. narrate.
- Day 21 was used as the end point of the experiment to detect the degree of fibrosis in the model.
- the total HYP content in the lung tissue of each test group was calculated, and the reduction ratio of HYP content in the peptide administration group relative to the model group was obtained to evaluate the inhibitory effect of the peptide molecules on collagen deposition in pathological fibrosis sites.
- the results are shown in Table 6.
- the HYP content in the polypeptide administration group was reduced to varying degrees compared with the model group, indicating that the polypeptide of the present invention inhibits collagen deposition in fibrotic tissue to varying degrees.
- Example 7 Inhibitory test of polypeptides on inflammation and fibrosis pathology in fibrosis mouse disease model
- This example is to evaluate the inhibitory effect of polypeptides on fibrosis pathology.
- Male C57/6J mice aged 6-8 weeks and 18-20g were used as the research subjects.
- the experimental animals were randomly divided into groups, with 6 mice in each group.
- the BLM model group and each treatment group were given a single tracheal dose of 0.7 USP/kg BLM solution for modeling, and the negative control group was given an equal volume of normal saline.
- Administration began on Day 8.
- the trachea of each peptide administration treatment group was administered 3 mg/kg peptide drug, and the positive control group was administered an equal dose of peptide SEQ ID NO: 50.
- the administration frequency was 2 times a week, with a total of 4 administrations;
- BLM model The experimental group and the negative control group were given an equal volume of solvent (physiological saline) as a control.
- the operation method is the same as described in Example 4.
- the lung tissue was dissected, washed in physiological saline to remove blood stains, and dried with gauze. Histopathological analysis was performed on the dissected lung tissue.
- the lung tissue was perfused with 0.5 mL of paraformaldehyde through the trachea and placed in 4 mL of paraformaldehyde. A small amount of gauze was put in to completely immerse the lung tissue in the fixative and fixed for more than 24 hours.
- One left lung lobe and four right lung lobes were trimmed to the largest coronal plane, placed in an embedding box, dehydrated overnight and then embedded in paraffin to make 3 ⁇ m sections.
- An automatic slide staining machine (Leica, model ST5010) was used to perform hematoxylin-Eosin (HE) staining (Hematoxylin-Eosin, HE) (Hematoxylin (Hutter, batch number 201905, batch number 201905), Cologne, batch number 2018070301), and MASSON'S trichrome method (celestite blue, Cologne, Batch number MKCH8129; Aniline blue, source leaf, lot number H70J11S115483; Ponceau red, Sigma, lot number SHBM2047) staining. After sealing, a digital panoramic scanner (Zhiyue, model WS-10) was used for scanning and processing.
- Each lung lobe was intercepted with ⁇ 10 fields of view under a 20x field of view, and the severity of inflammation and fibrosis were measured by HE staining and MASSON'S trichrome staining. Severity score of fibrosis is used to evaluate the inhibitory effect of peptides on inflammatory infiltration and fibrosis deposition in pathological sites of fibrosis. The results are shown in Figures 1 and 2.
- Figure 1 shows the HE stained pathological section (magnified 20 times).
- the lung tissue of the negative control group showed a vacuolar thin-walled structure in the alveolar cavity.
- BLM model group a large number of inflammatory cells and fibroblasts infiltrated in the interstitium and alveoli, accompanied by significant thickening of the alveolar interstitium and extrusion and deformation of the alveolar structure.
- Each peptide administration group showed varying degrees of reduction in inflammatory cells and reduction in alveolar septal thickening areas.
- Figure 2 shows the MASSON’S stained pathological section (magnification 20 times).
- the collagen layer of the bronchial wall of the mouse lung tissue was thin, and a small amount of strip-shaped and elongated collagen fibers could be seen between the alveoli.
- the alveolar intervals were widened, a large number of collagen fibers were deposited, and fibrotic masses formed in some areas.
- Each polypeptide administration group only had a small amount of alveolar septal widening, and the collagen fiber deposition area was reduced to varying degrees compared with the BLM model group.
- the results of inflammation score and fibrosis score are shown in Table 7. Compared with the negative control group, the inflammation score and fibrosis score of the BLM model group increased significantly, and the inflammation score and fibrosis score of each peptide administration group decreased to varying degrees compared with the model group.
- Example 8 Inhibitory test of collagen deposition by polypeptides in fibrosis rat disease model
- the total HYP content in the lung tissue of each test group was calculated, and the reduction ratio of HYP content in the peptide administration group relative to the model group was obtained to evaluate the inhibitory effect of the peptide molecules on collagen deposition in pathological fibrosis sites.
- the results are shown in Table 8.
- the HYP content in the polypeptide administration group was reduced to varying degrees compared with the model group, indicating that the polypeptide of the present invention has varying degrees of effects on collagen deposition in tissues at the pathological site of pulmonary fibrosis. of inhibition.
- Example 9 Inhibition test of polypeptides on fibrosis pathology in fibrosis rat disease model
- This example is to evaluate the inhibitory effect of polypeptide administration on the expression levels of profibrotic factors in pathological fibrosis sites.
- Male SD rats aged 6-8 weeks and 200-220g were used as the research subjects. The experiment was randomly divided into groups, with 6 rats in each group.
- Day 1 the BLM model group and each treatment group were given a single intratracheal dose of 1.8 USP/kg (100 ⁇ L) bleomycin solution to create the model, and the negative control group was given an equal volume of normal saline.
- Each peptide administration treatment group was given a 2 mg/kg dose of peptide into the trachea starting from Day 1.
- the administration frequency was twice a week, for a total of 4 administrations.
- the BLM model group and the negative control group were given an equal volume of solvent (physiological saline) as a For the control, the volume was all 100 ⁇ L, and the operation method was the same as described in Example 4.
- One left lung lobe and four right lung lobes were trimmed to the largest coronal plane.
- the left and right lungs were placed in two embedding boxes respectively, and 3 ⁇ m sections were made after paraffin embedding.
- MASSON'S staining was performed as described in Example 7. After digital scanning processing, ⁇ 10 fields of view were intercepted from each lung lobe under a 10x field of view, and the fibrosis severity was scored to evaluate the inhibitory effect of the polypeptide on fibrosis deposition in pathological fibrosis sites.
- the fibrosis score results are shown in Table 9. Compared with the negative control group, the lung coefficient and fibrosis severity score of the BLM model group increased significantly; the lung coefficient and fibrosis score of each peptide administration group decreased to varying degrees compared with the model group.
- TGF- ⁇ has dual roles in the tumor microenvironment (TME). In the early stages of tumors, the TGF- ⁇ signaling pathway induces apoptosis and inhibits tumor cell proliferation. In the late stage of tumors, TGF- ⁇ , as an immunosuppressive cytokine, inhibits the immune response and promotes tumor progression by regulating tumor cell movement and metastasis.
- TEM tumor microenvironment
- This example is to evaluate the effect of polypeptides on the migration ability of different tumor cells.
- the experiment uses a transwell nested chamber (membrane pore size is 8 ⁇ m).
- THP-1 cells are inoculated in the lower chamber and treated with 300 nM PMA (Sigma, Cat. No. P1585) for differentiation and attachment, and then add 100 ⁇ M peptide (an equal volume of solvent is added to the negative control group) for treatment 48 Hour.
- the polypeptide has a significant inhibitory effect on the migration ability of human lung adenocarcinoma cells, human liver cancer cells, and human breast cancer cells.
- Example 11 Inhibition of ECM-related gene expression by polypeptides in liver fibrosis cell model
- This example evaluates the inhibitory effect of polypeptides on the expression of extracellular matrix (ECM)-related genes in a liver fibrosis cell model.
- ECM extracellular matrix
- the experiment used human hepatic stellate cells LX-2. Hepatic stellate cells are the main source of myofibroblasts in the liver. They are activated and transformed into myofibroblasts, which secrete ECM in the liver and directly participate in the formation of liver fibrosis. Brief stimulation of LX-2 cells by TGF- ⁇ 1 can initiate positive feedback regulation of TSP-1-TGF- ⁇ 1 activation events and continuously promote ECM formation and other related myofibroblast characteristics.
- the experimental operation is as follows. After 20ng/mL TGF- ⁇ 1 (Genscript, Cat. No. Z03411) stimulated LX-2 cells (Procell, CL-0560) for 24 hours, 100 ⁇ M peptide was added and cultured for 48 hours. An equal volume of solvent was added to the model group, and LX without any treatment was added. -2 cells served as blank control. Afterwards, LX-2 cells were collected and total RNA was extracted for RT-qPCR to detect the expression of ECM-related genes, EDA-Fibronectin (FN) and Collagen I (COL1). The test results are shown in Table 11. The polypeptide can reduce the expression of FN and COL1 genes in LX-2 and inhibit the formation of ECM.
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Abstract
本发明涉及抑制TSP-1参与的TGF-β1活化的肽类抑制剂或其药学可接受的盐、溶剂化物或前药，以及包含所述肽或其药学可接受盐、溶剂化物或前药的药物组合物和应用其治疗或预防TGF-β1相关疾病，尤其是纤维化和实体瘤的方法。
Description
本发明涉及抑制TSP-1参与的TGF-β1活化的肽类抑制剂或其药学可接受的盐、溶剂化物或前药，以及包含所述肽或其药学可接受盐、溶剂化物或前药的药物组合物和应用其治疗或预防TGF-β1相关疾病，尤其是纤维化和实体瘤的方法。
纤维化是损伤修复过程中纤维结缔组织异常过度增生而形成组织瘢痕的病理过程，其特征在于细胞外基质(Extracellular Matrix,ECM)的过度沉积。纤维化的发生不仅会导致组织器官功能不可逆的衰退，严重时会影响患者生活质量，甚至危及生命。此外，纤维化的形成有利于肿瘤发生与远端转移，促进免疫抑制性的肿瘤微环境形成，引发免疫治疗耐受或免疫不应答。
在纤维化及肿瘤发生发展机制中，转化生长因子β(Transforming growth factorβ,TGFβ)是重要的核心调控分子。TGF-β及其信号通路已经被提出作为纤维化病症以及肿瘤的药物治疗靶点。然而，TGF-β作为多功能细胞因子，也参与多种正常生理过程，这导致了直接以TGF-β及其信号通路为靶点的药物具有较高的毒性。目前，一些此类药物开发项目在进入临床阶段后，已经因严重药物相关不良反应而进展缓慢，甚至终止。
TGF-β合成分泌时是非活性的前体结构，必须经过一系列的激活事件，成为活性片段后才能与其受体结合以介导下游通路转导。TGF-β的激活事件是在不同环境不同条件下，由不同的蛋白分子参与完成，因此以疾病情况下参与激活的特定蛋白为靶点已成为破解TGF-β成药困难的最新研究方向。
血小板反应蛋白-1(thrombospondin,TSP-1)是临床上已发现在纤维化，肿瘤相关适应症中才会活跃表达的蛋白。已有研究表明，TSP-1能够参与慢性炎症，纤维化过程中的TGF-β活化事件，是TGF-β1的体内主要激活剂(Cell,Vol.93,1159–1170,June 26,1998,Copyright1998by Cell Press)。考虑到临床应用，靶向TGF-β活化的肽类药物，由于其在TGF-β1被激活的部位起作用，并易于分解代谢和具有低抗原性，因此，可能提供比其他治疗性抗TGF-β1分子更安全的策略。因此，本发明拟针对TSP-1参与TGF-β活化的结构域进行多肽成分设计，以期提供改善纤维化和癌症的新肽类药物。
发明概述
通过锐意研究，本发明人意外地发现了一组包含氨基酸基序(DXED)的5-13个氨基酸的短肽，其不仅能够有效地抑制TSP-1受体与TSP-1/L-TGF-β1复合物的相互作用，且能有效阻断TSP-1依赖性的L-TGF-β1活化和后续的TGF-β信号传导。基于此，本发明人建立了本发明的抑制肽(下文也称作“TGF-β1活化抑制肽”)。如实施例所示，本发明的抑制肽，以环状和线性形式，均呈现出TGF-β1活化抑制活性；并且与现有技术中已知的类似肽(SEQ ID NO:50)相比，在缩短肽长度以实现更高成本效益的情况下，显示出了更强的TGF-β1活化抑制作用。在一些优选的实施方案中，与所述的已知类似肽相比，本发明的抑制肽
还呈现出更好的溶解度，稳定性和/或生物利用度。
此外，本发明的抑制肽也表现出有利的药物安全性优势。对TSP-1的研究显示，其包含多个功能结构域，这些结构域能够彼此独立地与不同细胞表面受体结合，参与止血、细胞黏附、细胞迁移、细胞凋亡以及抗血管生成等多种生理过程。本发明的抑制肽，由于分子量小、活性高、且针对TSP-1上参与TGF-β1活化的特定结构域而设计，因此，可以有效地降低对TSP-1的其余功能结构域及相关生理功能的干扰，保障了药物作用的特异性和安全性。
因此，在第一方面，本发明提供一种TGF-β1活化抑制肽或其药学上可接受的盐、溶剂化物或前药。根据本发明的TGF-β1活化抑制肽能够特异性阻断TSP-1参与的TGF-β1活化，并具有如下通式(I)的氨基酸序列：
R1-X1-X2-X3-X4-X5-X6-X7-R2 (I),
R1-X1-X2-X3-X4-X5-X6-X7-R2 (I),
其中，
R1是乙酰基或不存在；
R2为氨基或不存在；
X1不存在，或选自T和S；
X2选自天然氨基酸或非天然氨基酸，优选地，选自极性侧链氨基酸，例如不带电荷的极性侧链氨基酸S,C,G,N,Q,T,Y或带负电荷的极性侧链氨基酸D和E，更优选地选自氨基酸N，C，Q，S，T，E和D；更优选地选自氨基酸Q，C，N，E或S；
X3是D；
X4选自小侧链氨基酸，例如A，C，G，P，S，T和V，更优地选自氨基酸A，P和S，更优选地选自氨基酸A和P；
X5是E；
X6是D；
X7不存在，或是由选自氨基酸序列Z1Z2Z3SZ4LQ中依次从氨基端到羧基端的1个，2个，3个，4个，5个，6个或7个氨基酸组成的序列，
其中Z1是氨基酸N，Q、P、A、L、V、M、或I，优选N、P或L，最优选N或P；
Z2是氨基酸T、S、V、C、A、K、或R，优选T、C或K，更优选地，当Z2是氨基酸C或K时，Z2与X2或X3的氨基酸侧链形成共价连接，
Z3是氨基酸A、V、L、或I，优选A或V，
Z4是氨基酸F、Y、H、或W，优选F或H；
任选地，当X4为A时，在X3和X4之间插入0-1个小侧链氨基酸，例如P或G，优选地，插入P；
任选地，当X7由1-5个氨基酸组成时，所述肽在X7后具有的0-2个额外的氨基酸残基添加，例如，选自Q、E、D和N的氨基酸残基的1-2个氨基酸残基添加，或选自C或K的1个氨基酸残基添加。
在一些实施方案中，在本发明的肽中，
X1不存在，或选自T和S；
X2选自氨基酸Q，N，C，S，T，E和D；
X3是D；
X4选自A，C，G，P，S，T和V；
X5是E；
X6是D；
X7不存在，或是由选自氨基酸序列Z1Z2Z3SZ4LQ中依次从氨基端到羧基端的1个，2个，3个，4个，5个，6个或7个氨基酸组成的序列，其中，
Z1是N、P或L；
Z2是T或C或K，；
Z3是A或V；
Z4是F，Y，H，或W。
在一些实施方案中，在本发明的肽中，
X1选自T和S；
X2选自Q和N；
X3是D；
X4选自A和P；
X5是E；
X6是D。
在一些实施方案中，X7不存在。
在另一些实施方案中，X7由Z1、Z1Z2、Z1Z2Z3、Z1Z2Z3S、Z1Z2Z3SZ4、Z1Z2Z3SZ4L或Z1Z2Z3SZ4LQ组成，优选地，X7由Z1Z2Z3、Z1Z2Z3S或Z1Z2Z3SZ4组成，
其中，优选地
Z1是N、P或L，优选N或P；
Z2是T、K或C；
Z3是A或V，优选A；
Z4是F，Y，H，或W，优选F或H。
本发明的肽可以是线性肽或环化肽。
在第二方面，本发明也提供了，本发明的抑制肽或其药学上可接受的盐、溶剂化物或前药在TSP-1参与的TGF-β1活化所致的疾病或与之相关的疾病(即，“TGF-β1相关疾病”)中的治疗和/或预防性应用。本发明也提供本发明抑制肽或其药学上可接受的盐、溶剂化物或前药应用于制备预防或治疗纤维化病症的药物。所述的纤维化病症以TSP-1参与的TGF-β1激活为特征，表现出细胞外基质(Extracellular Matrix,ECM)的过度沉积，但不限于疾病发生的部位，也不限于传统的疾病分类。本发明也提供本发明抑制肽应用于制备预防或治疗与TGF-β1活化相关的癌症的药物。
附图简述
图1显示，纤维化小鼠疾病模型的HE染色病理切片。通过HE染色进行炎症严重程度评估，显示本发明多肽对纤维化病理部位炎性浸润的抑制效果。
图2显示，纤维化小鼠疾病模型的MASSON’S染色病理切片。通过MASSON’S三色法染色，进行纤维化严重程度评估，显示本发明多肽对纤维化病理部位纤维化沉积的抑制效果。
图3显示，纤维化大鼠疾病模型的MASSON’S染色病理切片，显示本发明多肽对纤维化病理部位纤维化沉积的抑制效果。
发明内容
定义
为了可以更容易地理解本发明，某些科技术语具体定义如下。除非本文其它部分另有明确定义，否则本文所用的科技术语都具有本发明所属领域普通技术人员通常理解的含义。氨基酸残基的缩写是本领域中所用的指代20个常用氨基酸之一的标准3字母和/或1字母代码。除非另外指明，否则氨基酸序列以氨基到羧基的方向从左到右书写。
本文(包括权利要求书)所用单数形式包括其相应的复数形式，除非文中另有明确规定。
术语“约”是指，在本领域普通技术人员所确定的特定值的可接受误差范围内的值，所述误差范围取决于测量或确定所述值时使用的测量手段，即测量系统的限制。比如，“约”可以是指根据本领域中的实践在1个或大于1个标准偏差内。或者，“约”可以是指多达5％、10％或20％的范围(即，±5％、±10％或±20％)。
术语“和/或”当用于连接两个或多个可选项时，应理解为意指可选项中的任一项或可选项中的任意两项或更多项。
如本文中所用，术语“包含”或“包括”意指包括所述的要素、整数或步骤，但是不排除任意其他要素、整数或步骤。在本文中，当使用术语“包含”或“包括”时，除非另有指明，否则也涵盖由所述及的要素、整数或步骤组成的情形。例如，当提及“包含”某个具体序列时，也旨在涵盖由该具体序列组成的肽或多肽。
如本文所用，术语“肽”和“多肽”可互换使用，是指长度介于2个与100个氨基酸之间的氨基酸序列，这些氨基酸通过肽键连接。氨基酸可以是天然存在的和非天然存在的。
如本文中使用的，术语“保守氨基酸取代”或“保守氨基酸替代”意指，不会不利地影响或改变包含氨基酸序列的多肽的生物学功能的氨基酸取代。典型的保守型氨基酸取代是指将一种氨基酸取代为具有相似的化学性质(例如电荷或疏水性)的另一种氨基酸。功能上相似氨基酸的保守性置换表是本领域熟知的。在本发明中，保守取代残基可以来自以下的保守替代表，尤其是表中的优选保守氨基酸取代残基。
发明详述
在研究中，本发明人令人惊奇地发现，具有富含负电荷的氨基酸基序(DXED)的短肽能够有效地引发对TSP-1依赖性TGF-β1活化的抑制效应。通过构效关系和氨基酸取代等分析，本发明人进一步识别了该肽序列中的必需氨基酸和可能取代的位点；并通过肽环化和/或主链修饰，进行结构优化，建立了一系列具有改善的生物活性和/或物化性质的新型TGF-β1活化抑制肽。
本发明的抑制肽
在一方面，因此，本发明提供一种TGF-β1活化抑制肽或其药学上可接受的盐、溶剂化物或前药。本发明肽包含富含负电荷的核心基序(DXED)，并具有5-13个氨基酸长。优选地，本发明肽由7-11个氨基酸组成。在一些实施方案中，本发明肽是线性或环化五肽或六肽，例如线性或首位环化的六肽。在一些实施方案中，本发明肽是线性或环化的七肽，例如首尾环化的七肽。在另一些实施方案中，本发明肽是线性或环化的八肽，尤其是侧链成环的八肽，优选地，二硫键成环。在另一些实施方案中，本发明肽是线性或环化的九肽，尤其是线性九肽。在另一些实施方案中，本发明肽是线性或环化的十肽，例如首尾环化的十肽。在另一些实施方案中，本发明肽是线性或环化的十一肽、十二肽或十二肽，优选线性十一肽。
在一些实施方案中，本发明肽与序列TQDAEDNTVSFLQ(SEQ ID NO:51)通过比对以最大对齐后，在本发明肽的全长和与之对齐的SEQ ID NO:51相应部分之间，具有0-5个氨基酸差异，例如0-2个氨基酸的差异。所述氨基酸差异包括氨基酸替代、添加和缺失。例如，本发明肽可以与序列SEQ ID NO:51在相应部分相同，或具有1或2个氨基酸替代、或具有N端或C端，尤其是C端的1-2个氨基酸添加。用于替代的氨基酸可以是天然氨基酸或非天然氨基酸。在一些实施方案中，本发明肽包含一个脯氨酸替代，优选地位于核心序列DXED的第二个残基位置；或位于紧靠核心序列C端的第一个残基位置。在一些实施方案中，本发明肽的总净电荷为负，例如，-1，-2，-3或-4，优选地-2或-3。
在一些实施方案中，所述肽包含选自以下的核心氨基酸序列：DAED或DPED。在另一些实施方案中，所述肽还包含位于所述核心序列C端的N或P残基，由此，所述肽包含选自以下的氨基酸序列：DAEDN；DPEDN；或DAEDP。
在一些实施方案中，本发明肽为环肽。在一些实施方案中，本发明肽可以通过首尾氨基酸的共价连接而环化。在另一些实施方案中，本发明肽可以通过两个氨基酸侧链之间形成的内酰胺键或二硫键来环化。为此，可以将带有适当侧链基团的适宜氨基酸或者类似物置于本发明肽的合适位置中，以促成肽的分子内酰胺键或二硫键形成。在一些实施方案中，内酰胺通过位于本发明肽的核心基序(DXED)C-端的氨基酸残基的侧链氨基官能团，与位于核心基序(DXED)N-端的氨基酸的羧酸基团偶联形成。在一些实施方案中，二硫键通过氧化偶联位于核心基序(DXED)C-端的半胱氨酸与位于核心基序(DXED)N-端的半胱氨酸形成。在内酰胺环肽的一些实施方案中，核心基序的第一个天冬氨酸(D)或位于其N端的天冬氨基酸(D)或谷氨酸(E)的侧链羧基，与位于核心基序C端的赖氨酸(K)的侧链氨基形成内酰胺键，且成环的两个氨基酸之间相隔不少于4个氨基酸，例如，相隔4个、5个或6个氨基酸，优选地，成环的赖氨酸残基是所述肽的C末端最后一个残基。在二硫键成环的环肽的一些实施方案中，紧靠核心基序(DXED)N-端的第一个氨基酸为半胱氨酸，与位于核心基序(DXED)C-端的半胱氨酸形成二硫键，优选地，成环的两个氨基酸之间相隔不少于5个氨基酸，优选地，相隔5个或6个氨基酸，再优选地，成环的C-端半胱氨酸是所述肽的C末端最后一个残基。在一些实施方案中，本发明的肽优选为二硫键成环的环肽，且优选地由8个氨基酸组成，更优选地，所述环肽的N末端氨基被乙酰化和/或C末端羧基被酰胺化。
本发明的肽可以包含化学修饰，例如，N端乙酰化，C端酰胺化，PEG修饰，脂质修饰，D型氨基酸替代、或非天然氨基酸替代。在一些实施方案中，本发明肽通过与允许肽保持其TGF-β1活化抑制能力的分子共价连接来修饰，包括例如，糖基化、乙酰化、聚乙二醇化、磷酸化、酰胺化或利用已知保护/封闭基团衍生化。在一个实施方案中，修饰是N末端酰化(尤其是乙酰化)。在一个实施方案中，修饰是C末端酰胺化。优选地，本发明的肽为N末端酰化(尤其是乙酰化)的线性肽或侧链成环的环肽，再优选地，所述肽还具有C末端酰胺化。在一些实施方案中，肽可以连接到生物分子或者用于结合、标记或鉴定的材料上。
根据本发明的TGF-β1活化抑制肽能够抑制TSP-1依赖性的TGF-β1活化，且优选地具有至少如下至少一项性质：
-降低纤维化组织中的活性TGF-β1量；
-抑制TGF-β1介导的下游信号传导；
-减少纤维化组织中胶原沉积；
-抑制由TGF-β1介导的炎性和/或纤维化病变；
-抑制由TGF-β1刺激的胞外基质相关基因表达；
-抑制肿瘤细胞迁移；
-预防或治疗TGF-β1相关疾病，尤其是纤维化或癌症。本领域技术人员可以根据本领域已知的或实施例中所述的方法测定本发明肽的上述性质。
在一些实施方案中，本发明的肽包含对应于SEQ ID NOs:1-49中的一者的氨基酸序列或由其组成。在一些实施方案中，本发明肽包含SEQ ID NOs:1-49中的一者或由其组成。在一些实施方案中，本发明的肽包含对应于SEQ ID NOs:6,14-15,22-26,33,36,40,43-46和48-49中的一者的氨基酸序列或由其组成。在一些实施方案中，本发明的肽包含SEQ ID NOs:6,14-15,22-26,33,36,40,43-46和48-49中的一者或由其组成。优选地，本发明的肽在如实施例2的测定中阻断TSP-1与其受体的结合。在一些实施方案中，在如实施例2的测定中，本发明肽表现出高于15％、20％、25％、30％、35％、40％或更高的阻断率。
在一些实施方案中，本发明的肽包含对应于SEQ ID NOs:3-6,14,17,22,24-26,31,33,36,40-47中的一者的氨基酸序列或由其组成。在一些实施方案中，本发明肽包含SEQ ID NOs:3-6,14,17,22,24-26,31,33,36,40-47中的一者或由其组成。在一些实施方案中，本发明的肽包含对应于SEQ ID NOs:3,6,14,22,24-26,33,41-46中的一者的氨基酸序列或由其组成。在一些实施方案中，本发明的肽包含SEQ ID NOs:3,6,14,22,24-26,33,41-46中的一者或由其组成。优选，本发明的肽在如实施例3的测定中抑制TSP-1依赖性TGF-β1活化，且优选地相对于无多肽加入的阴性对照，使TGF-β1活化降低达到25％,30％,35％或以上。
在一些实施方案中，本发明的肽包含对应于SEQ ID NOs:6,22,25-26,33,40,43,44,46中的一者的氨基酸序列或由其组成。在一些实施方案中，本发明的肽包含SEQ ID NOs:6,22,25-26,33,40,43,44,46中的一者或由其组成。在一些实施方案中，本发明的肽包含SEQ ID NO:6或由其组成。在一些实施方案中，本发明的肽包含SEQ ID NO:22或由其组成。在一些实施方案中，本发明的肽包含SEQ ID NO:25或由其组成。在一些实施方案中，本发明的肽包含SEQ ID NO:26或由其组成。在一些实施方案中，本发明的肽包含SEQ ID NO:33或由其组成。在一些实施方案中，本发明的肽包含SEQ ID NO:43或由其组成。
以下为本发明TGF-β1活化抑制肽的一些实施方案。
1.一种具有如下通式(I)的氨基酸序列的肽或其药学上可接受的盐、溶剂化物或前药：
R1-X1-X2-X3-X4-X5-X6-X7-R2 (I),
R1-X1-X2-X3-X4-X5-X6-X7-R2 (I),
其中，
R1为乙酰基或不存在；
X1不存在，或为天然或非天然的氨基酸残基；
X2和X4各自独立地为天然或非天然的氨基酸残基；
X3是D；
X5是E；
X6是D；
X7是0-7个氨基酸组成的序列；且
R2为氨基或不存在。
2.根据实施方案1的肽或其药学上可接受的盐、溶剂化物或前药，其中，
X1不存在，或选自高丝氨酸,别苏氨酸,T和S；
X2选自天然氨基酸或非天然氨基酸，优选地，选自极性侧链氨基酸，例如不带电荷的极性侧链氨基酸S,C,G,N,Q,T,Y或带负电荷的极性侧链氨基酸D和E，更优选地选自氨基酸N，C，Q，S，T，E和D；更优选地选自氨基酸Q，C，N，E或S；
X3是D；
X4选自小侧链氨基酸，例如A，C，G，P，S，T和V，更优地选自氨基酸A，P和S，更优选地选自氨基酸A和P；
X5是E；
X6是D；
X7不存在，或是由选自氨基酸序列Z1Z2Z3SZ4LQ中依次从氨基端到羧基端的1个，2个，3个，4个，5个，6个或7个氨基酸组成的序列，
其中，Z1,Z2,Z3,Z4彼此独立地选自天然氨基酸和非天然氨基酸，优选地：
其中Z1是氨基酸N，Q、P、A、L、V、M、或I，优选N、P或L，最优选N或P；
Z2是氨基酸T、S、V、C、A、K、或R，优选T、C或K，更优选地，当Z2是氨基酸C或K时，Z2与X2或X3的氨基酸侧链形成共价连接，
Z3是氨基酸A、V、L、或I，优选A或V，
Z4是氨基酸F、Y、H、或W，优选F或H；
任选地，当X4为A时，在X3和X4之间插入0-1个小侧链氨基酸，例如P或G，优选地，插入P；
任选地，当X7由1-5个氨基酸组成时，所述肽在X7后具有的0-2个额外的氨基酸残基添加，例如，彼此独立地选自Q、E、D和N的1个或2个氨基酸残基添加，或选自C或K的1个氨基酸残基添加。
3.根据实施方案1-2的肽或其药学上可接受的盐、溶剂化物或前药，其中，X1不存在。
4.根据实施方案1-2的肽或其药学上可接受的盐、溶剂化物或前药，其中，X1是T或S。
5.根据实施方案1-2的肽或其药学上可接受的盐、溶剂化物或前药，其中，X1是T。
6.根据实施方案1-5的肽或其药学上可接受的盐、溶剂化物或前药，其中，X2选自极性侧链氨基酸。
7.根据实施方案1-5的肽或其药学上可接受的盐、溶剂化物或前药，其中，X2选自不带电荷的极性侧链氨基酸S,C,G,N,Q,T和Y。
8.根据实施方案1-5的肽或其药学上可接受的盐、溶剂化物或前药，其中，X2选自带负电荷的极性侧链氨基酸D和E。
9.根据实施方案1-5的肽或其药学上可接受的盐、溶剂化物或前药，其中，X2选自氨基酸N，C，高半胱氨酸,Q，S，高丝氨酸,T，别苏氨酸,E和D。
10.根据实施方案1-5的肽或其药学上可接受的盐、溶剂化物或前药，其中，X2选自氨基酸Q，C，N，E或S。
11.根据实施方案1-5的肽或其药学上可接受的盐、溶剂化物或前药，其中，X2为Q。
12.根据实施方案1-5的肽或其药学上可接受的盐、溶剂化物或前药，其中，X2为C。
13.根据实施方案1-5的肽或其药学上可接受的盐、溶剂化物或前药，其中，X2的侧链与包含在X7中的氨基酸的侧链形成共价连接，优选地，X2和Z2均为C且两者的侧链巯基形成二硫键。
14.根据实施方案1-13的肽或其药学上可接受的盐、溶剂化物或前药，其中，X4为小侧链氨基酸，例如A，C，G，P，S，T和V。
15.根据实施方案1-13的肽或其药学上可接受的盐、溶剂化物或前药，其中，X4选自氨基酸A，P和S。
16.根据实施方案1-13的肽或其药学上可接受的盐、溶剂化物或前药，其中，X4为A。
17.根据实施方案1-13的肽或其药学上可接受的盐、溶剂化物或前药，其中，X4为P。
18.根据实施方案1-17的肽或其药学上可接受的盐、溶剂化物或前药，其中，X7不存在。
19.根据实施方案1-17的肽或其药学上可接受的盐、溶剂化物或前药，其中，X7为由选自氨基酸序列Z1Z2Z3SZ4LQ中依次从氨基端到羧基端的1个，2个，3个，4个，5个，6个或7个氨基酸组成的序列。
20.根据实施方案1-17的肽或其药学上可接受的盐、溶剂化物或前药，其中，X7为由选自氨基酸序列Z1Z2Z3SZ4中依次从氨基端到羧基端的1个，2个，3个，4个或5个氨基酸组成的序列。
21.根据实施方案1-17的肽或其药学上可接受的盐、溶剂化物或前药，其中，X7由Z1组成。
22.根据实施方案1-17的肽或其药学上可接受的盐、溶剂化物或前药，其中，X7由Z1Z2组成。
23.根据实施方案1-17的肽或其药学上可接受的盐、溶剂化物或前药，其中，X7由Z1Z2Z3组成。
24.根据实施方案1-17的肽或其药学上可接受的盐、溶剂化物或前药，其中，X7由Z1Z2Z3S组成。
25.根据实施方案1-17的肽或其药学上可接受的盐、溶剂化物或前药，其中，X7由Z1Z2Z3S Z4组成。
26.根据实施方案1-17的肽或其药学上可接受的盐、溶剂化物或前药，其中，X7由Z1Z2Z3S Z4L组成。
27.根据实施方案1-17的肽或其药学上可接受的盐、溶剂化物或前药，其中，X7由Z1Z2Z3S Z4LQ组成。
28.根据实施方案1-27的肽或其药学上可接受的盐、溶剂化物或前药，其中，Z1是N，Q、P、A、N-甲基丙氨酸、L、正亮氨酸、V、M、N-甲基甲硫氨酸、或I。
29.根据实施方案1-27的肽或其药学上可接受的盐、溶剂化物或前药，其中，Z1是N。
30.根据实施方案1-27的肽或其药学上可接受的盐、溶剂化物或前药，其中，Z1是P。
31.根据实施方案1-27的肽或其药学上可接受的盐、溶剂化物或前药，其中，Z1是L。
32.根据实施方案1-31的肽或其药学上可接受的盐、溶剂化物或前药，其中，Z2是T、别苏氨酸、S、高丝氨酸、V、C、高半胱氨酸、A、N-甲基丙氨酸、K、或R。
33.根据实施方案1-31的肽或其药学上可接受的盐、溶剂化物或前药，其中，Z2是T。
34.根据实施方案1-31的肽或其药学上可接受的盐、溶剂化物或前药，其中，Z2是C，且与X2通过侧链共价连接。
35.根据实施方案1-31的肽或其药学上可接受的盐、溶剂化物或前药，其中，Z2是K，且与X3通过侧链共价连接。
36.根据实施方案1-35的肽或其药学上可接受的盐、溶剂化物或前药，其中，Z3是A、N-甲基丙氨酸、V、L、正亮氨酸、或I。
37.根据实施方案1-35的肽或其药学上可接受的盐、溶剂化物或前药，其中，Z3是A，尤其是在Z1是N或P时。
38.根据实施方案1-35的肽或其药学上可接受的盐、溶剂化物或前药，其中，Z3是V。
39.根据实施方案1-38的肽或其药学上可接受的盐、溶剂化物或前药，其中，Z4是芳族氨基酸。
40.根据实施方案1-38的肽或其药学上可接受的盐、溶剂化物或前药，其中，Z4是F，N-甲基苯丙氨酸、高苯丙氨酸、Y，H，或W。
41.根据实施方案1-38的肽或其药学上可接受的盐、溶剂化物或前药，其中，Z4是F或H。
42.根据实施方案1-38的肽或其药学上可接受的盐、溶剂化物或前药，其中，Z4是H。
43.根据实施方案1-42的肽或其药学上可接受的盐、溶剂化物或前药，其中，X7由Z1组成；或由Z1Z2组成；或由Z1Z2Z3组成；或由Z1Z2Z3S组成；或由Z1Z2Z3SZ4组成。
44.根据实施方案1-43的肽或其药学上可接受的盐、溶剂化物或前药，其中，X7为选自以下的氨基酸序列：N；P；L；NTA；NTV；PTA；NTAS；PTAS；NTVSF；NTASF；NTASH；NTVSFLQ。
45.根据实施方案1-44的肽或其药学上可接受的盐、溶剂化物或前药，其中，所述肽在X7后具有0-2个额外的氨基酸残基添加。
46.根据实施方案1-45的肽或其药学上可接受的盐、溶剂化物或前药，其中，所述肽包含选自以下的氨基酸序列：DAED；DPED；DAEDN；DPEDN；或DAEDP。
47.根据实施方案1-46的肽或其药学上可接受的盐、溶剂化物或前药，其中，X1为T且X2为Q。
48.根据实施方案1-47的肽或其药学上可接受的盐、溶剂化物或前药，其中，R1为乙酰基。
49.根据实施方案1-48的肽或其药学上可接受的盐、溶剂化物或前药，其中，R2为氨基。
50.一种具有如下通式(II)的氨基酸序列肽或其药学上可接受的盐、溶剂化物或前药：
R1-X1-X2-D-X4-E-D-X7-R2 (II),
R1-X1-X2-D-X4-E-D-X7-R2 (II),
其中，
R1为乙酰基或不存在；
R2为氨基或不存在；
X1为T或S；
X2选自极性侧链氨基酸；
X4选自氨基酸A或P；
X7是由选自氨基酸序列Z1TZ3SZ4中依次从氨基端到羧基端的1个，2个，3个，4个或5个氨基酸组成的序列，其中，
其中Z1是N、P或L；
Z3是A、V、L、或I，
Z4是F、Y、H、或W，优选F或H。
51.根据实施方案50的肽或其药学上可接受的盐、溶剂化物或前药，其中，X2选自氨基酸Q，N，C，S，T，E和D；优选选自Q，N，E或S；更优选X2是Q或N。
52.根据实施方案50的肽或其药学上可接受的盐、溶剂化物或前药，其中，X2是Q。
53.根据实施方案50-52的肽或其药学上可接受的盐、溶剂化物或前药，其中，Z1是N且Z3是A或V；或Z1是P且Z3是A。
54.根据实施方案50-53的肽或其药学上可接受的盐、溶剂化物或前药，其中，Z4是F或H。
55.一种具有如下通式(III)的氨基酸序列肽或其药学上可接受的盐、溶剂化物或前药：
R1-X1-环(-C-D-X4-E-D-Z1-C)-X8-R2 (III),
R1-X1-环(-C-D-X4-E-D-Z1-C)-X8-R2 (III),
其中，
R1为乙酰基或不存在；
R2为氨基或不存在；
X1为T或S；
X4选自小侧链氨基酸，例如A，C，G，P，S，T和V，更优地选自氨基酸A，P和S，更优选地选自氨基酸A和P；
Z1是天然氨基酸或非天然氨基酸；
X8不存在，或是1-5个氨基酸组成的序列。
56.根据实施方案55的肽或其药学上可接受的盐、溶剂化物或前药，其中，X4是A，C，G，P，S，T或V。
57.根据实施方案55的肽或其药学上可接受的盐、溶剂化物或前药，其中，X4是A。
58.根据实施方案55的肽或其药学上可接受的盐、溶剂化物或前药，其中，X4是P。
59.根据实施方案55-58的肽或其药学上可接受的盐、溶剂化物或前药，其中，Z1是极性不带电荷氨基酸或非极性氨基酸。
60.根据实施方案55-58的肽或其药学上可接受的盐、溶剂化物或前药，其中，Z1是N，Q、P、A、L、V、M、或I。
61.根据实施方案55-58的肽或其药学上可接受的盐、溶剂化物或前药，其中，Z1是N。
62.根据实施方案55-61的肽或其药学上可接受的盐、溶剂化物或前药，其中，X8不存在。
63.根据实施方案55-61的肽或其药学上可接受的盐、溶剂化物或前药，其中，X8由选自氨基酸序列Z3SZ4LQ中依次从氨基端到羧基端的1个，2个,3个,4个或5个氨基酸组成的序列，其中，Z3是A、V、L、或I，且Z4是F、Y、H、或W，优选F或H。
64.根据实施方案63的肽或其药学上可接受的盐、溶剂化物或前药，其中，Z3是A或V，且Z4是F或H。
65.根据实施方案55-64的肽或其药学上可接受的盐、溶剂化物或前药，其中，所述肽由5至13个氨基酸组成，优选由7、8、9、10或11个氨基酸组成。
66.根据实施方案1-54的肽或其药学上可接受的盐、溶剂化物或前药，其中，所述肽由5至13个氨基酸组成，优选由7、8、9、10或11个氨基酸组成。
67.根据实施方案66的肽或其药学上可接受的盐、溶剂化物或前药，其中，所述肽为线性肽。
68.根据实施方案66的肽或其药学上可接受的盐、溶剂化物或前药，其中，所述肽为环肽，且优选地，所述肽的环化区域包含核心基序(DXED)。
69.根据实施方案68的肽或其药学上可接受的盐、溶剂化物或前药，其中，所述肽为首尾成环或侧链成环。
70.根据实施方案68的肽或其药学上可接受的盐、溶剂化物或前药，其中，所述肽为首尾成环。
71.根据实施方案68的肽或其药学上可接受的盐、溶剂化物或前药，其中，所述肽为侧链成环。
72.根据实施方案68的肽或其药学上可接受的盐、溶剂化物或前药，其中，所述肽通过第3位和第8位氨基酸的侧链偶联成环、或通过第2位和第8位氨基酸的侧链偶联成环。
73.根据实施方案71-72的肽或其药学上可接受的盐、溶剂化物或前药，其中，所述肽通过氨基酸侧链之间形成的内酰胺键环化。
74.根据实施方案71-72的肽或其药学上可接受的盐、溶剂化物或前药，其中，所述肽通过氨基酸侧链之间形成的二硫键环化。
75.根据实施方案71-74的肽或其药学上可接受的盐、溶剂化物或前药，其中，所述肽通过侧链成环，
其中，X3是D，Z2是K，且所述肽通过X3与Z2的侧链之间形成的内酰胺键环化；或
其中，X2是C，Z2是C，且所述肽通过X2与Z2的侧链之间形成的二硫键环化，
优选地，当Z2是氨基酸C或K时，X7由Z1Z2组成，且X1为T或S。
76.一种肽或其药学上可接受的盐、溶剂化物或前药，其中，所述肽具有对应于选自SEQ ID NO：1至49之任一的氨基酸序列、或包含选自SEQ ID NO：1至49之任一，或由其组成。
77.一种肽或其药学上可接受的盐、溶剂化物或前药，其中，所述肽具有对应于选自SEQ ID NO：6,22,25,26,33,40,43,44和46之任一的氨基酸序列、或包含选自SEQ ID NO：6,22,25,26,33,40,43,44和46之任一。
78.一种肽或其药学上可接受的盐、溶剂化物或前药，其中，所述肽包含SEQ ID NO：6或由其组成。
79.一种肽或其药学上可接受的盐、溶剂化物或前药，其中，所述肽包含SEQ ID NO：22或由其组成。
80.一种肽或其药学上可接受的盐、溶剂化物或前药，其中，所述肽包含SEQ ID NO：25或由其组成。
81.一种肽或其药学上可接受的盐、溶剂化物或前药，其中，所述肽包含SEQ ID NO：26或由其组成。
82.一种肽或其药学上可接受的盐、溶剂化物或前药，其中，所述肽包含SEQ ID NO：33或由其组成。
83.一种肽或其药学上可接受的盐、溶剂化物或前药，其中，所述肽包含SEQ ID NO：40或由其组成。
84.一种肽或其药学上可接受的盐、溶剂化物或前药，其中，所述肽包含SEQ ID NO：43或由其组成。
85.一种肽或其药学上可接受的盐、溶剂化物或前药，其中，所述肽包含SEQ ID NO：44或由其组成。
86.一种肽或其药学上可接受的盐、溶剂化物或前药，其中，所述肽包含SEQ ID NO：46或由其组成。
87.一种肽或其药学上可接受的盐、溶剂化物或前药，其中，所述肽与根据实施方案76-86之一的肽相比，具有1、2、3、4或5个氨基酸差异，优选地，1个或2个氨基酸替代、缺失或添加，优选地，所述替代发生在核心基序(DXED)之外的氨基酸残基上，例如为保守氨基酸替代。
88.根据实施方案1-87的肽或其药学上可接受的盐、溶剂化物或前药，其中，所述肽是化学修饰的。
89.根据实施方案1-88的肽或其药学上可接受的盐、溶剂化物或前药，其中，所述肽包含PEG修饰。
90.根据实施方案1-89的肽或其药学上可接受的盐、溶剂化物或前药，其中，所述肽包含脂质修饰。
91.根据实施方案1-90的肽或其药学上可接受的盐、溶剂化物或前药，其中，所述肽包含D型氨基酸替代和/或非天然氨基酸替代。
本发明抑制肽的制备方法
在一个方面，本发明提供用于产生本发明抑制肽的方法。可以使用化学合成法、发酵、或基因重组技术进行本发明肽的生产。
化学合成肽的方法为本领域熟知，包括固相合成和液相合成技术。可以使用Merrifield开发的固相多肽合成(SPPS)技术，合成期望的肽链。也可以使用自动肽合成仪，进行合成。相比于重组合成的肽，SPPS肽产品的后续纯化更简单和容易。因此，应用SPPS技术来合成本发明的短肽，是优选的。
SPPS技术一般由偶联和脱保护循环组成。在Fmoc合成策略中，在偶联步骤中，应用缩合剂例如HBTU/HATU/DIC等，Fmoc保护的氨基酸(Fmoc-AA-OH)被偶联到固体聚合物树脂上；在脱保护步骤中，通过脱保护剂例如哌啶等，保护基Fmoc自氨基酸上去除，以释放氨基，从而进入下一个偶联和脱保护循环。整个过程可以在筛式反应器中进行，直到最终的肽合成并从树脂上切割下来。之后，可以通过高效液相色谱纯化和检测合成的肽产品。
有各种树脂，例如4-甲苯氢胺(HMBA)树脂、Wang树脂、2-氯三苯甲基氯(CTC)树脂和Merrifield树脂，可以用于肽的固相合成。此外，通过将树脂与不同的接头偶联，也开发了多种功能性树脂，以允许在固相中进行肽环化。
在一个实施方案中，本发明提供一种用于本发明肽的制备方法，所述方法包括：(1)合成具有规定序列的线性肽，例如，以4-甲苯氢胺(HMBA)树脂为起始原料，以Fmoc保护氨基酸为单体，六氢吡啶/DMF溶液为脱保护试剂，在缩合剂和碱性条件作用下，依次逐个接上氨基酸，以产生所述的线性肽；(2)可选地，对步骤(1)中得到的线性肽进行环化；(3)纯化步骤(1)或(2)得到的肽产品。
在另一个实施方案中，本发明提供一种用于本发明肽的制备方法，所述方法包括：(1)重组表达具有规定序列的线性肽；(2)可选地，对步骤(1)中得到的线性肽进行环化；(3)纯化步骤(1)或(2)得到的肽产品。在一些方面，本发明也提供了包含编码本发明肽的氨基酸序列的核酸、包含所述核酸的载体(例如表达载体)和宿主细胞。
肽环化是一种常见的肽修饰技术，可以包括各种策略，例如首尾环化、侧链到侧链的环化、以及主链到侧链的环化。通常，在不与其他肽连接的情况下，单个线性肽过于灵活。而环化通过组织分子内相互
作用，可以促进肽的二级结构形成，从而提高肽的稳定性。
在根据本发明的一个实施方案中，本发明的肽通过首尾环化形成单环肽。在根据本发明的另一实施方案中，本发明的肽，通过侧链成环，在谷氨酸(E)或天冬氨酸(D)和赖氨酸(K)的侧链之间形成内酰胺桥。在根据本发明的另一实施方案中，本发明的肽，通过侧链成环，在两个半胱氨酸(C)残基的侧链之间形成二硫键。在包含内酰胺桥的本发明环肽实施方案中，优选地，赖氨基酸残基位于本发明的核心序列(DXED)的C端，并与核心序列(DXED)的第一个天冬氨酸D残基形成所述的内酰胺桥，更优选地，所述K残基与D残基之间相隔至少4个残基，例如，4,5，或6个残基，优选地所述赖氨酸残基为本发明肽的C末端最后一个残基。在包含二硫键的本发明环肽实施方案中，优选地，用于成环的两个半胱氨酸残基分别位于核心序列(DXED)的N端和C端，更优选地，两个半胱氨酸残基之间相隔至少5个残基，例如，5,6,7,8个残基。在一个优选实施方案中，本发明肽的第3位氨基酸是D且第8位氨基酸是K，且本发明肽通过两者的侧链形成内酰胺键而成环。在另一个优选实施方案中，本发明肽的第2位氨基酸是C且第8位氨基酸是C，且本发明肽通过两者的侧链形成二硫键而成环。
肽合成后，可以使用或不使用医学化学技术，对其进行修饰。在一些情况下，修饰肽可能是有利的，例如，通过修饰以模拟、稳定或构建一个更合适的二级结构，以改善肽药物的生物学活性、和/或改善肽药物的选择性、稳定性和溶解度。肽修饰可以是对非必要氨基酸的氨基酸替换或残基修饰，和/或对肽的N端和/或C端的修饰，例如N端乙酰化修饰和C端酰胺化修饰。修饰也可以是对影响肽生物活性的关键氨基酸残基的替代，以寻求活性的改变。
在一些情况下，肽可以包含非天然氨基酸。根据需要，肽可以包含1个、2个、3个、4个、5个、或6个、或更多个非天然氨基酸。或者，组成肽的氨基酸可以各自独立地选自天然氨基酸。
化学合成的肽往往携带游离的氨基和游离的羧基。在一些情况下，可以对肽的末端进行封闭，即N端乙酰化和C端酰胺化，使合成肽更接近天然蛋白质的模拟物，并提高肽的稳定性。
因此，在一些方面，本发明的肽可以包含D-氨基酸、非天然氨基酸、氨基酸类似物、和/或基团取代和修饰；或可以与聚合物或与药物载体连接。
药物组合物
在一方面，本发明也提供了包含本发明肽或其药学可接受盐、溶剂化物或前药的药物组合物。如本领域中明了的，药物组合物还可以任选地包含合适的药用辅料，药用载体、药用赋形剂，包括缓冲剂。
用于肽给药的施用途径包括，但不限于，皮下、肌内和静脉给药，粘膜给药(例如，鼻腔给药、肺粘膜给药、舌下给药)、口服给药(例如，加入胃肠道促渗剂或载体)、和透皮给药。可以将肽配制为适于给药的任何形式，如注射剂，例如静脉注射剂或静脉滴注药，冻干粉等。用于不同制剂形式的辅料是本领域已知的。
如本领域明了的，药物组合物还可以包含对待治疗的具体疾病而言有益的其它治疗剂。在一些实施方案中，因此，本发明也提供药物组合物，其包含本发明肽或其药学可接受盐、溶剂化物或前药，并还包含对待治疗的具体疾病而言有益的其它治疗剂。在这样的实施方案中，本发明的肽可以与所述其它治疗剂包
含在相同或不同的组合物中；并且可以与所述其它治疗剂同时、相继、或以任何顺序和任何给药方案进行给药。
在一些实施方案中，包含在本发明药物组合物中尤其可以是前述实施方案1-91的本发明肽或其药学可接受的盐，尤其是前述实施方案76-86之一的本发明肽或其药学可接受的盐，尤其是，前述实施方案78-86之一的本发明肽或其药学可接受的盐。
治疗方法和用途
在一个方面，本发明提供本发明的肽或其药学可接受盐、溶剂化物或前药或本发明的药物组合物用作药物，用于在受试者中预防或治疗TGF-β相关疾病的用途、和相应的疾病治疗和预防方法。
本文术语“TGF-β相关疾病”是指，与TSP-1参与的TGF-β1活化或TGF-β1活性的病理性增加相关、和/或由其所导致的疾病和病症，包括但不限于纤维化、慢性炎症、和癌症。所述的TGF-β相关疾病将受益于对TSP-1参与的TGF-β1活化的抑制作用。
在本文中，术语“受试者”或“患者”或“个体”包括任何人或非人动物。术语“非人动物”包括所有脊椎动物，例如哺乳动物和非哺乳动物，诸如非人灵长类动物、绵羊、狗、猫、马、牛、鸡、两栖动物、爬行动物等。优选地，本发明的受试者是人。
在本文中，术语“有效量”或“有效剂量”是指本发明的肽或其药学上可接受盐、溶剂化物或前药，当单独或与其它治疗药物组合，给予细胞、组织或受试者时，有效预防或改善一种或多种疾病或病况的症状或该疾病或病况的发展的量。有效剂量还指足以导致症状改善的量，例如治疗、治愈、预防或改善相关医学病况或者提高这类病况的治疗、治愈、预防或改善的速度的量。当对个体施用单独给予的活性成分时，有效剂量仅是指该成分。当组合施用时，有效剂量是指引起治疗或预防效果的活性成分的综合量，不论是组合、依次给予还是同时给予。在一些实施方案中，有效量将导致诊断标准或参数的至少10％，通常至少20％，优选至少约30％，更优选至少40％，最优选至少50％改善。
在根据本发明的一些实施方案中，疾病或病症的“治疗”是指改善疾病或病症(即，减缓或阻止或减少疾病的进展或其临床症状的至少一个)。在另一些实施方案中，“治疗”是指缓解或改善至少一个身体参数，包括可能不能被患者辨别出的那些生理参数。在另一些实施方案中，“治疗”是指在身体上(例如，可辨别的症状的稳定)、生理上(例如，身体参数的稳定)或在这两方面调节疾病或病症。除非在本文中明确描述，否则用于评估疾病的治疗和/或预防的方法在本领域中通常是已知的。
在根据本发明的再一些实施方案中，疾病或病症的“预防”包括对疾病或病症或特定疾病或病症的症状的发生或发展的抑制。通常，在纤维化或癌症的背景中，术语“预防”是指在纤维化或癌症的病征或症状发生前，特别是在具有发病风险的受试者中进行的药物施用。
因此，在一些实施方案中，本发明提供预防或治疗TGF-β相关疾病的方法，包括向有需要的受试者施用有效量的根据本发明的肽或其药学上可接受的盐、溶剂化物或前药或根据本发明的药物组合物。任选地，所述方法进一步包含向所述受试者施用有效量的第二治疗剂，例如化学治疗剂或免疫治疗剂。优选地，根据本发明方法治疗的所述TGF-β相关疾病，具有病变部位的巨噬细胞浸润。在一些实施方案中，所述TGF-
β相关疾病与组织损伤、炎症或纤维化相关。在一些实施方案中，所述TGF-β相关疾病涉及肿瘤细胞的转移。
在一些实施方案中，在本发明的预防或治疗方法中，尤其可以应用前述实施方案1-91的本发明肽或其药学可接受的盐，尤其是前述实施方案76-86之一的本发明肽或其药学可接受的盐，尤其是，前述实施方案78-86之一的本发明肽或其药学可接受的盐。
纤维化病症治疗
在一些实施方案中，与TGF-β活性的病理性增加相关的病症是纤维化。在一个实施方案中，本发明提供本发明肽或其药学可接受盐、溶剂化物或前药或本发明药物组合物用作药物，用于在受试者中预防或治疗纤维化病症的用途、和相应的治疗和预防方法。
目前，已知的具有纤维化特征的疾病，涉及非常庞大的疾病谱，可以根据其发生的组织器官进行分类，如肺、肝、肾、骨髓等组织的纤维化；也可以将其归属于常见的疾病分类，如慢性炎症类疾病，肿瘤类疾病，免疫类疾病等。具有纤维化这一病理特征，正在作为重新分类甚至命名、诊断、治疗纤维化病症的重要依据，而不再受限于对于疾病发生部位的认知，或局限于传统的疾病分类。
在本文中，因此，“纤维化病症”是指，具有纤维化病理表型特征的疾病，而不限于疾病发生的具体部位或疾病的传统分类。可用本发明方法治疗的纤维化疾病的实例包括，但不限于，各种组织器官的纤维化，例如，肺纤维化、肝纤维化、肾纤维化；以及慢性炎症、肿瘤，和免疫疾病。
在一些实施方案中，因此，本发明提供用于在受试者中预防或治疗纤维化病症的方法，包括向有需要的受试者施用预防或治疗有效量的根据本发明的肽或其药学上可接受的盐、溶剂化物或前药或根据本发明的药物组合物。在一些实施方案中，所述纤维化病症是肝纤维化、肺纤维化、肾纤维化、骨髓纤维化、皮肤纤维化、或心脏纤维化。
在一些实施方案中，所述纤维化病症是肺纤维化，例如特发性肺纤维化(IPF)。在一些实施方案中，该方法包括通过吸入向受试者施用本发明肽，例如，作为雾化制剂。
本文所述的治疗用途和方法还可以包括共同配制和/或共同施用本发明的肽或其药学可接受盐、溶剂化物或前药或药物组合物与另一种治疗有效的药剂以预防和/或治疗病理性纤维化疾病。
肿瘤治疗
在一些实施方案中，与TGF-β活性的病理性增加相关的病症是癌症。在一个实施方案中，本发明提供本发明肽或其药学可接受盐、溶剂化物或前药或药物组合物用作药物，用于在受试者中预防或治疗癌症的用途、和相应的治疗和预防方法。在一些实施方案中，所述癌症是实体瘤，优选地选自：肺癌、肝癌、乳腺癌、子宫癌、前列腺癌、胰腺癌、结肠癌、皮肤癌、中枢神经系统癌症、纤维肌瘤、纤维瘤、纤维腺瘤和纤维肉瘤。在一些实施方案中，癌症是肉瘤、胰腺癌、胶质母细胞瘤、头颈癌、黑色素瘤、乳腺癌或结肠直肠癌。在一些实施方案中，癌症选自鳞状细胞癌、表皮样癌、尿路上皮癌、腺癌、肾上腺皮质癌、基底细胞癌、导管原位癌(DCIS)、浸润性导管癌、胸腺癌和肾细胞癌。
在一些实施方案中，该方法包括通过口服、静脉内、瘤内、透皮、皮下或局部施用途径，向受试者施用本发明的肽或其药学可接受盐、溶剂化物或前药或药物组合物。在一些实施方案中，该方法包括使受试者的癌性组织(例如癌性皮肤组织)与含有治疗有效量的本发明肽或其药学可接受盐、溶剂化物或前药或药物组合物的制剂接触足以治疗癌症的时间。
本文所述的治疗用途和方法还可以包括共同配制和/或共同施用本发明的肽或其药学可接受盐、溶剂化物或前药或药物组合物与另一种治疗有效的药剂，以预防和/或治疗癌症。
其他应用
在一些方面，本发明也提供了本发明肽或其药学可接受盐、溶剂化物或前药的如下应用：
-阻断CD36与TSP-1的结合；
-抑制TSP-1依赖性的TGF-β1活化；
-降低纤维化组织中的活性TGF-β1量；
-抑制TGF-β1介导的下游信号传导；
-减少纤维化组织中胶原沉积；
-抑制由TGF-β1介导的炎性和/或纤维化病变；
-抑制由TGF-β1刺激的胞外基质相关基因表达；
-抑制肿瘤细胞迁移；
-预防或治疗TGF-β1相关疾病，尤其是纤维化或癌症；
或用于制备在上述用途中使用的药物。
上述应用可以是体外的或体内的。在优选的实施方案中，所述应用为体内的疾病治疗性或预防性应用。在所述的体内应用方法中，包括以治疗有效量施用本发明肽或其药学可接受盐、溶剂化物或前药，任选地联合其它治疗剂。
为了预防或治疗疾病，本发明肽或其药学可接受盐、溶剂化物或前药的合适剂量(当单独或与一种或多种其他的治疗剂组合使用时)将取决于待治疗疾病的类型、具体药物的类型、疾病的严重性和进程、以预防目的施用还是以治疗目的施用、以前的治疗、患者的临床病史和对所述药物的应答，和主治医师的判断。所述药物可以以一次治疗或经过一系列治疗合适地施用于患者。
再一方面，本发明也提供本发明肽或其药学可接受盐、溶剂化物或前药在制备用于前述方法(例如用于治疗)的药物中的用途。
下面为了辅助对本发明的理解，进一步给出如下实施例。然而，应当理解，这些实施例不意在且不应当以任何方式构成对本发明的保护范围的限制。
为了本发明的目的，在本文提及氨基酸残基时，适用于以下的氨基酸代码，包括三字母和单字母代码：
实施例1多肽制备
本发明实施例所使用的多肽采用Fmoc(9-芴甲氧羰基)固相合成法制备。以线性肽羧基端氨基酸为合成起点，在多肽固相合成反应柱中，首先将Rink Amide MBHA Resin树脂(吉尔生化，49101)在DCM溶液中浸泡30min进行树脂活化，之后抽干，加入20％的六氢吡啶+80％的DMF溶液，通氮气搅拌鼓动30min，进行脱保护反应；投料Fmoc保护氨基酸、缩合剂及有机碱进行反应；反应后使用茚三酮显色法检测反应是否完全。按照每条肽(从羧基端到氨基端)的氨基酸顺序，不断重复上述投料、反应、检测步骤，直到最后一个氨基酸，之后将目的肽从树脂上切割得到粗品肽。
环化多肽操作步骤如下：
(1)二硫键成环：将按前述在树脂上合成并切割下来的线性多肽，在pH＝7.5-8的水溶液中(肽浓度1g/L)，搅拌12h以上，进行成环。
(2)侧链成环：在前述的线性多肽合成过程中，选取特殊的侧链保护基，如选择D(oall)和K(alloc)，保护D的侧链羧基和K的侧链氨基。在线性多肽序列合成结束后，在树脂上去除-oall/-alloc，再通过相应缩合剂进行成环。
(3)首尾成环：将按前述在树脂上合成并切割下来的线性多肽做成全保护的多肽片段，在DMF或者其他溶剂中加入相应缩合剂，进行成环。反应结束后，产物自水中析出、烘干、切割环肽上的保护基，得到去保护的首尾环化多肽粗品。
合成的线性和环状粗品肽通过HPLC分离纯化，其中选择C18柱，0.1％TFA/乙腈溶液的洗脱液A，0.1％TFA/水溶液的洗脱液B。收集主峰，经质谱检测产品的分子量和理论值相吻合，冻干后即得到精品肽。合成的多肽序列如表1所示，其中每个序列编号代表的肽由序列描述中的“序列”部分和“修饰”部分共同组成。
表1多肽序列表
实施例2多肽靶标体外阻断活性试验
ELISA法检测多肽对TSP-1蛋白与TSP-1受体蛋白CD36结合的阻断能力。使用100mM碳酸盐缓冲液将TSP-1蛋白(novoprotein，货号CU45)稀释至2μg/mL，并加入至96孔酶标板(Thermo，货号437111)样品孔(100μL/孔)中，4℃孵育过夜；使用1×PBST洗液洗板2次后，每孔加入200μL 1.5％BSA(Sigma，货号B2064)，37℃孵育2h；用1×PBST洗液洗板3次后，加入分析物(100μL/孔，3个平行孔，对照组加入0.25μg/mL的Fc标签的CD36蛋白(R&D Systems，货号1955-CD-050)；多肽处理组加入终浓度为0.25μg/mL的Fc标签的CD36蛋白与100μM多肽的混合体系)，37℃孵育3h；用1×PBST洗液洗板5次后，加入FITC标记的抗Fc标签抗体(碧云天，货号A0556)的1：500稀释液，37℃孵育1h；用1×PBST洗液洗板5次后，使用多功能酶标仪(TECAN，型号Infinite 200Pro)检测各样品孔中FITC荧光信号强度。通过计算多肽处理组相较对照组的荧光信号比值，得出多肽阻断CD36与TSP-1蛋白结合的阻断率，即，(对照组的荧光信号-多肽处理组的荧光信号)/对照组的荧光信号×100％，评估多肽阻断CD36与TSP-1蛋白结合的能力(表2-1)。基于单浓度阻断率结果和多肽分子特性，优选多肽分子进行浓度(0μM、0.5μM、1μM、5μM、10μM、100μM)依赖性试验，测定多肽阻断CD36与TSP-1蛋白结合的IC50值，进
一步评估优选多肽分子阻断CD36与TSP-1结合的能力(表2-2)。试验结果显示，本发明多肽均能够抑制CD36与TSP-1的结合。
表2-1多肽的靶标体外结合试验数据
表2-2多肽阻断CD36与TSP-1蛋白结合的IC50值
实施例3多肽在细胞模型中对TSP-1依赖的TGF-β活化的抑制试验
本实施例通过TSP-1高表达的细胞模型，检测多肽对于TSP-1依赖的TGF-β活化的抑制作用。试验采用佛波酯(Phorbol 12-myristate 13-acetate，PMA)(Sigma，货号P1585)诱导THP-1细胞(美国典型培养物保藏中心，ATCC，TIB-202)分化为具有明显的巨噬细胞表达特征的巨噬细胞样细胞。在该细胞模型中TSP-1蛋白表达显著增加，并分泌L-TGF-β；在无干扰L-TGF-β活化的因子存在时，细胞分泌的纤溶酶将识别并切割与细胞表面CD36受体结合的TSP-1/L-TGF-β复合物，从而产生增加量的活性TGF-β。
试验操作如下：96孔板中接种THP-1细胞并经300nM PMA分化贴壁处理，形成巨噬细胞样细胞。加入100μM多肽(阴性对照组加入等体积多肽溶剂PBS，阳性对照组加入等浓度多肽SEQ ID NO：50)，处理24小时后，接种用于检测TGF-β活性的293报告基因细胞株，293-TGFβRes细胞株(novoprotein，货号XCC03-1)共培养24小时。加入Bio-Lite Luciferase Assay含荧光底物的裂解液(Vazyme，货号DD1201-01)，使用多功能酶标仪(TECAN，型号Infinite 200Pro)检测萤光强度。通过计算多肽处理组相较阴性对照组的萤光信号比值，评估多肽对TGF-β活化的抑制作用。试验结果如表3所示，在该细胞模型中，多肽对TGF-β活性有不同程度的抑制效果，表明本发明的多肽能够抑制TSP-1依赖的TGF-β活化。
表3多肽在细胞模型中对TSP-1依赖的TGFβ活化抑制作用
实施例4纤维化小鼠疾病模型中多肽对活性TGFβ含量的抑制试验
本实施例为评估多肽对纤维化疾病进展过程中活性TGF-β含量的抑制效果。本实施例采用博来霉素(Bleomycin，BLM)诱导的小鼠肺纤维化模型。BLM动物模型具有与原发病病理特征相似度高、操作简便、重复性好等优点，是最具代表性的纤维化机制模型，也是药效研究公认推荐使用的动物模型。
本实施例以6-8周龄18-20g雄性C57/6J小鼠为研究对象，实验动物采用随机分组，每组8只，实验终点(Day8)检测该模型中促纤维化因子表达情况。本实施例中的造模和给药方法均使用气溶胶气管内定量给药器(玉研，型号YAN30012)进行气管内给药。给药操作如下(An Official American Thoracic Society Workshop Report:Use of Animal Models for the Preclinical Assessment of Potential Therapies for Pulmonary Fibrosis,Am J Respir Cell Mol Biol Vol 56,Iss 5,pp 667–679,May 2017)：采用小动物麻醉机(瑞沃德，型号R500IE)将异氟烷(瑞沃德，批号22041701)雾化后麻醉实验动物。取麻醉状态适宜的动物，使动物头部向上，身体倾斜仰卧，并通过门齿悬挂于操作平台上(操作平台倾斜约60°)。术者右手持组织镊轻轻夹起舌头向外上方推动并打开动物下颌，左手持喉镜(玉研，型号SR310-RW)，将喉镜叶形前端叶片沿舌体正中滑进口腔至声门前并向上轻轻压住舌体和舌根，将给药器针头从口腔贴着喉镜插入到动物声门内，快速推注给药器中50μL液体。造模当日(Day1)将实验动物随机分入各组，通过气管给予0.7USP/kg剂量博来霉素溶液(瀚晖制药，批号20080511)进行造模；Day7时各多肽给药治疗组单次气管内给予50μL多肽药物(3mg/kg剂量)，模型组给予等体积的溶剂(生理盐水，科伦，批号L221062501)作为对照；给药24小时后解剖动物取肺组织，经液氮研磨后提取小鼠肺组织总蛋白，使用TGF-βELISA检测试剂盒(BOSTER，货号EK0515)测定各组小鼠肺组织中活性TGF-β含量，计算多肽给药组与模型组中活性TGF-β含量的比值，评估多肽对活性TGF-β含量的影响。结果如表4所示，在纤维化疾病模型中，多肽给药组相比模型组，活性TGF-β含量有不同程度降低，表明本发明多肽对纤维化进展过程中由肺损伤诱导的活性TGF-β含量增加具有抑制作用。
表4多肽对纤维化小鼠模型中活性TGF-β含量的抑制
实施例5纤维化小鼠疾病模型中多肽对TGFβ下游信号通路的抑制试验
本实施例为评估多肽对纤维化疾病进展过程中TGF-β下游信号通路转导的抑制作用。TGF-β胞内信号转导依赖于Smad家族蛋白的磷酸化事件，其中Smad2/3磷酸化含量的增加是判断TGF-β信号通路转导发生的重要标志。
本实施例以6-8周龄18-20g雄性C57/6J小鼠为研究对象，实验动物采用随机分组，每组8只。本实施例中的造模和给药方法均使用气溶胶气管内定量给药器进行气管内给药，操作方法同实施例4描述。造模当日(Day1)将实验动物随机分入各组，通过气管给予0.7USP/kg剂量博来霉素溶液(瀚晖制药，批号20080511)进行造模；Day7时各多肽给药治疗组单次气管内给予3mg/kg剂量多肽药物，BLM模型组给予等体积的溶剂(生理盐水)作为对照；给药24小时后解剖动物取肺组织冻存于液氮中备用；液氮研磨后
提取小鼠肺组织总蛋白，使用P-Smad2/3ELISA检测试剂盒(CST，货号12001C)测定总组织中P-Smad2/3含量，计算多肽给药组与模型组P-Smad2/3含量的比值，评估多肽对TGF-β信号通路转导的影响。结果如表5所示，在纤维化疾病模型中，多肽给药组相比模型组，P-Smad2/3蛋白含量有不同程度的降低，表明本发明多肽对纤维化进展过程中TGF-β信号通路转导有不同程度的抑制。
表5多肽对纤维化小鼠模型中TGF-β信号通路转导的抑制
实施例6纤维化小鼠疾病模型中多肽对胶原沉积的抑制试验
本实施例为评估多肽对纤维化组织中胶原沉积的抑制作用。本实施例以6-8周龄18-20g雄性C57/6J小鼠为研究对象，实验动物采用随机分组，每组8只。造模当日(Day1)，单次气管给予0.7USP/kg剂量博来霉素溶液造模；Day8开始治疗给药，各多肽给药治疗组气管给予3mg/kg剂量多肽药物，给药频率每周2次，共计给药4次，BLM模型组给予等体积的溶剂(生理盐水)作为对照，操作方法同实施例4描
述。Day21作为实验终点，检测该模型中的纤维化程度。
Day21解剖动物取肺组织，经液氮研磨后用酸水解法提取小鼠肺组织中羟脯氨酸(hydroxyproline，HYP)含量。HYP是胶原中一种特有的氨基酸，HYP含量的测定是反映胶原组织代谢及纤维化程度的一项重要指标。每100mg组织中加入1mL 6N HCl充分混匀，121℃水解6h，离心取水解液上清或不同浓度梯度的HYP标准品(Sigma，货号V900395-25G)溶液，用于HYP含量检测。96孔板中，每孔加入10μL水解液上清，置于65℃烘箱中烘干；每孔加入100μL 1.4％氯胺T(Sigma，货号23270-50G)氧化溶液，室温孵育15min；每孔加入100μL Ehrlich显色液(索莱宝，货号G1290)，65℃孵育30min；使用多功能酶标仪(TECAN，型号Infinite 200Pro)检测各样品孔在560nm下的吸光值(OD560)。计算各试验组肺组织中总HYP含量，得出多肽给药组相对于模型组的HYP含量减少比例，评估多肽分子对纤维化病理部位胶原沉积的抑制效果。结果如表6所示，在纤维化疾病模型中，多肽给药组相比模型组，HYP含量有不同程度的降低，表明本发明多肽对纤维化组织中胶原沉积有不同程度的抑制。
表6多肽对纤维化组织中胶原蛋白沉积的抑制
实施例7纤维化小鼠疾病模型中多肽对炎性及纤维化病理的抑制试验
本实施例为评估多肽对纤维化病理的抑制作用。以6-8周龄18-20g雄性C57/6J小鼠为研究对象，实验动物采用随机分组，每组6只。造模当日(Day1)，BLM模型组与各治疗组单次气管给予0.7USP/kg剂量BLM溶液造模，阴性对照组给予等体积生理盐水。Day8开始给药，各多肽给药治疗组气管给予3mg/kg剂量多肽药物，阳性对照组给予等剂量的多肽SEQ ID NO：50，给药频率每周2次，共计给药4次；BLM模型组及阴性对照组给予等体积的溶剂(生理盐水)作为对照。操作方法同实施例4描述。
Day21解剖取肺组织，生理盐水中清洗去除血污，纱布蘸干后，对解剖的肺组织进行组织病理分析。肺组织经气管灌注0.5mL多聚甲醛并置于4mL多聚甲醛中，放入少量纱布使肺组织完全浸入固定液中，固定24小时以上。将左侧1个肺叶和右侧肺4个肺叶均修剪出最大冠状面，放入一个包埋盒，脱水过夜后进行石蜡包埋制作3μm切片。自动载片染色机(Leica，型号ST5010)进行苏木素(赫特，批号201905)-伊红(科隆，批号2018070301)(Hematoxylin-Eosin，HE)染色，和MASSON’S三色法(天青石蓝，科隆，批号MKCH8129；苯胺蓝，源叶，批号H70J11S115483；丽春红，Sigma，批号SHBM2047)染色。
封片后使用数字化全景扫描仪(智跃，型号WS-10)扫描处理，每个肺叶于20倍视野下截取≤10个视野，通过HE染色和MASSON’S三色法染色分别进行炎症严重程度和纤维化严重程度评分，评估多肽对纤维化病理部位炎性浸润和纤维化沉积的抑制效果。结果显示在图1和图2中。
图1为HE染色病理切片(放大20倍)。阴性对照组肺组织为肺泡腔呈空泡状薄壁结构。BLM模型组在间质和肺泡内出现大量炎性细胞和成纤维细胞浸润，伴随肺泡间质明显增厚、肺泡结构被挤压变形。各多肽给药组均出现不同程度的炎性细胞减少，肺泡间隔增厚区域减少的情况。
图2为MASSON’S染色病理切片(放大20倍)。阴性对照组中，小鼠肺组织支气管壁胶原层较薄，肺泡间可见少量、条状、细长的胶原纤维分布。BLM模型组中，肺泡间隔增宽，可见大量胶原纤维沉积，部分区域形成纤维化团块。各多肽给药组仅有少量肺泡间隔增宽，胶原纤维沉积面积较BLM模型组出现不同程度的减小。
炎性评分和纤维化评分结果如表7所示。与阴性对照组相比，BLM模型组的炎性评分和纤维化评分均有明显增加，各多肽给药组的炎性评分和纤维化评分相比模型组均出现不同程度的下降。
表7多肽对小鼠纤维化模型中炎性及纤维化病理的抑制
实施例8纤维化大鼠疾病模型中多肽对胶原蛋白沉积的抑制试验
本实施例为评估多肽对肺纤维化病理部位胶原蛋白沉积的抑制作用。以6-8周龄200-220g的雄性SD大鼠为研究对象，实验采用随机分组，每组8只。造模当日(Day1)，模型组与各治疗组单次气管内给予1.8USP/kg剂量博来霉素溶液造模；各多肽给药治疗组于Day1起开始气管给予2mg/kg剂量多肽，给药频率每周2次，共计给药4次，模型组给予等体积的溶剂(生理盐水)作为对照，体积均为100μL，操作方法同实施例4描述。Day15作为实验终点，检测该模型中的纤维化程度。
Day15解剖取肺组织。经液氮研磨后用酸水解法提取大鼠肺组织中总羟脯氨酸(HYP)，利用氯胺T氧化显色法测定HYP含量。每100mg组织中加入1mL 6N HCl充分混匀，121℃水解6h，离心取水解液上清，用于HYP含量检测。96孔板中，每孔加入10μL水解液上清或不同浓度梯度的HYP标准品(Sigma，货号V900395-25G)溶液，置于65℃烘箱中烘干；每孔加入100μL 1.4％氯胺T(Sigma，货号23270-50G)氧化溶液，室温孵育15min；每孔加入100μL Ehrlich显色液(索莱宝，货号G1290)，65℃孵育30min；
使用多功能酶标仪(TECAN，型号Infinite 200Pro)检测各样品孔在560nm下的吸光值(OD560)。计算各试验组肺组织中总HYP含量，得出多肽给药组相对于模型组的HYP含量减少比例，评估多肽分子对纤维化病理部位胶原蛋白沉积的抑制效果。结果如表8所示，在肺纤维化疾病模型中，多肽给药组相比模型组，HYP含量有不同程度的降低，表明本发明多肽对肺纤维化病理部位组织中胶原蛋白沉积有不同程度的抑制。
表8：多肽对纤维化模型中胶原蛋白沉积的抑制
实施例9：纤维化大鼠疾病模型中多肽对纤维化病理的抑制试验
本实施例为评估多肽给药对纤维化病理部位促纤维化因子表达水平的抑制效果。以6-8周龄200-220g的雄性SD大鼠为研究对象，实验采用随机分组，每组6只。造模当日(Day1)，BLM模型组与各治疗组单次气管内给予1.8USP/kg剂量(100μL)博来霉素溶液造模，阴性对照组给予等体积生理盐水。各多肽给药治疗组于Day1起开始气管给予2mg/kg剂量多肽，给药频率每周2次，共计给药4次，BLM模型组及阴性对照组均给予等体积的溶剂(生理盐水)作为对照，体积均为100μL，操作方法同实施例4描述。
Day15解剖取肺组织，生理盐水中清洗去除血污，纱布蘸干后称肺重，计算肺系数(肺系数＝肺湿重/体重*1000，肺系数反映肺组织中的水肿、炎症和纤维化等大体情况)。对解剖的肺组织进行组织病理分析。肺组织经气管灌注5mL多聚甲醛并置于40mL多聚甲醛中，放入少量纱布使肺组织完全浸入固定液中，固定24h以上。将左侧1个肺叶和右侧肺4个肺叶均修剪出最大冠状面，左、右肺分别放入两个包埋盒，石蜡包埋后制作3μm切片。按实施例7中描述的方法进行MASSON’S染色。数字化扫描处理后，每个肺叶于10倍视野下截取≤10个视野，进行纤维化严重程度评分，以评估多肽对纤维化病理部位纤维化沉积的抑制效果。
结果如图3所示(放大10倍)。阴性对照组中，大鼠肺组织支气管壁胶原层较薄，肺泡间可见少量、条状、细长的胶原纤维分布。BLM模型组中，肺组织肺泡间隔增宽，可见大量胶原纤维沉积，部分区域形成纤维化团块。各多肽给药组有少量肺泡间隔增宽，胶原纤维沉积面积较BLM模型组出现不同程度的减小。
纤维化评分结果见表9。与阴性对照组相比，BLM模型组的肺系数、纤维化严重程度评分明显增加；各多肽给药组的肺系数和纤维化评分相比模型组均出现不同程度的下降。
表9多肽对大鼠纤维化模型中纤维化病理的抑制
实施例10多肽对肿瘤细胞迁移能力的抑制试验
TGF-β在肿瘤微环境(TME)中具有双重作用。在肿瘤早期，TGF-β信号通路诱导细胞凋亡，抑制肿瘤细胞增殖。在肿瘤晚期，TGF-β作为一种免疫抑制细胞因子对免疫应答产生抑制作用，并通过调节肿瘤细胞运动和转移等，促进肿瘤进展。
本实施例为评估多肽对不同肿瘤细胞迁移能力的影响。试验使用transwell嵌套室(膜孔径为8μm)，在下室接种THP-1细胞并经300nM PMA(Sigma，货号P1585)分化贴壁处理，再加入100μM多肽(阴性对照组加入等体积溶剂)处理48小时。上室接种肿瘤细胞人肺腺癌细胞A549细胞(美国典型培养物保藏中心，ATCC，CCL-185)共培养24小时，或者接种人肝癌细胞HepG2/C3A细胞(ATCC，CRL-10741)共培养24小时，或者接种人乳腺癌细胞MCF7细胞(ATCC，HTB-22)共培养48小时。之后，取上室，70％乙醇固定10min后清洗，再使用吉姆萨染液(碧云天，货号C0131)染色45min，双蒸水清洗2-3次后，用棉棒擦除上层未迁移的细胞，待小室干燥后在显微镜下观察，100倍镜下随机选取不同视野5张进行计数统计。结果如表10所示，多肽对人肺腺癌细胞、人肝癌细胞、人乳腺癌细胞的迁移能力有显著的抑制作用。
表10多肽对肿瘤细胞迁移能力的抑制作用
实施例11肝纤维化细胞模型中多肽对ECM相关基因表达的抑制
本实施例为评估多肽对肝纤维化细胞模型中胞外基质(ECM)相关基因表达的抑制作用。试验采用人肝星状细胞LX-2。肝星状细胞是肝脏中的肌成纤维细胞的主要来源，其被激活后转化为肌成纤维细胞，在肝脏中分泌ECM，直接参与肝纤维化形成。TGF-β1短暂刺激LX-2细胞能够启动TSP-1-TGF-β1活化事件的正反馈调节，不断促进ECM形成等相关肌成纤维细胞特征。
实验操作如下。20ng/mL TGF-β1(金斯瑞，货号Z03411)刺激LX-2细胞(Procell，CL-0560)24小时后，加入100μM多肽培养48小时，模型组加入等体积溶剂，未经任何处理的LX-2细胞作为空白对照。之后，收集LX-2细胞并提取总RNA进行RT-qPCR，以检测ECM相关基因，纤连蛋白EDA-Fibronectin(FN)和胶原蛋白Collagen I(COL1)的表达情况。试验检测结果如表11所示，多肽能够降低LX-2中FN与COL1基因的表达，抑制ECM形成。
表11肝纤维化细胞模型中多肽对ECM相关基因表达的抑制作用
序列表概述
本申请附带有序列表。下表提供了有关序列表中每个SEQ ID NO所描述的肽的信息，其中Ac表示乙酰基，NH2表示氨基。
Claims (31)
- 一种具有式(I)的氨基酸序列的肽或其药学上可接受的盐、溶剂化物或前药，
R1-X1-X2-X3-X4-X5-X6-X7-R2 (I),其中，R1为乙酰基或不存在；R2为氨基或不存在；X1不存在，或选自T和S；X2选自天然氨基酸或非天然氨基酸，优选地，选自极性侧链氨基酸，例如不带电荷的极性侧链氨基酸S,C,G,N,Q,T,Y或带负电荷的极性侧链氨基酸D和E，更优选地选自氨基酸N，C，Q，S，T，E和D；更优选地选自氨基酸Q，C，N，E或S；X3是D；X4选自小侧链氨基酸，例如A，C，G，P，S，T和V，更优地选自氨基酸A，P和S，更优选地选自氨基酸A和P；X5是E；X6是D；X7不存在，或是由选自氨基酸序列Z1Z2Z3SZ4LQ中依次从氨基端到羧基端的1个，2个，3个，4个，5个，6个或7个氨基酸组成的序列，其中Z1是氨基酸N，Q、P、A、L、V、M、或I，优选N、P或L，最优选N或P；Z2是氨基酸T、S、V、C、A、K、或R，优选T、C或K，更优选地，当Z2是氨基酸C或K时，Z2与X2或X3的氨基酸侧链形成共价连接，Z3是氨基酸A、V、L、或I，优选A或V，Z4是氨基酸F、Y、H、或W，优选F或H；任选地，当X4为A时，在X3和X4之间插入0-1个小侧链氨基酸，例如P或G，优选地，插入P；任选地，当X7由1-5个氨基酸组成时，所述肽在X7后具有0-2个额外的氨基酸残基添加，例如，选自Q、E、D和N的氨基酸残基的1-2个氨基酸残基添加，或选自C或K的1个氨基酸残基添加。 - 根据权利要求1的肽或其药学上可接受的盐、溶剂化物或前药，其中，X1不存在，或选自T和S；X2选自氨基酸Q，N，C，S，T，E和D；X3是D；X4选自A，C，G，P，S，T和V；X5是E；X6是D；X7不存在，或是由选自氨基酸序列Z1Z2Z3SZ4LQ中依次从氨基端到羧基端的1个，2个，3个，4个，5个，6个或7个氨基酸组成的序列，其中，Z1是N，Q、P、A、L、V、M、或I；Z2是T、K或C；Z3是A或V；Z4是F，Y，H，或W。
- 根据权利要求1-2任一项的肽或其药学上可接受的盐、溶剂化物或前药，其中，X1选自T和S；X2选自Q、N、E、S或C；X3是D；X4选自A和P；X5是E；X6是D。
- 根据权利要求1-3任一项的肽或其药学上可接受的盐、溶剂化物或前药，其中，所述肽包含选自以下的氨基酸序列：-DAED；-DPED；-DAEDN；-DPEDN；或-DAEDP。
- 根据权利要求1-4任一项的肽或其药学上可接受的盐、溶剂化物或前药，其中，X7由Z1、Z1Z2、Z1Z2Z3、Z1Z2Z3S、Z1Z2Z3SZ4、Z1Z2Z3SZ4L或Z1Z2Z3SZ4LQ组成，优选地，X7由Z1Z2Z3、Z1Z2Z3S或Z1Z2Z3SZ4组成，且其中Z1是N、P或L；Z2是T、K或C；Z3是A或V；Z4是F，Y，H，或W。
- 根据权利要求5的肽或其药学上可接受的盐、溶剂化物或前药，其中，Z1是N或P；Z2是T、K或C；Z3是A或V，优选A；Z4是F或H。
- 根据权利要求1-6任一项的肽或其药学上可接受的盐、溶剂化物或前药，其中，X7为选自以下的氨基酸序列：N；P；L；NTA；NTV；PTA；NTAS；PTAS；NTVSF；NTASF；NTASH；NTVSFLQ。
- 根据权利要求1-7任一项的肽或其药学上可接受的盐、溶剂化物或前药，其中，X1是T。
- 根据权利要求1-8任一项的肽或其药学上可接受的盐、溶剂化物或前药，其中，X4是A。
- 根据权利要求1-8任一项的肽或其药学上可接受的盐、溶剂化物或前药，其中，X4是P。
- 根据权利要求1-10任一项的肽或其药学上可接受的盐、溶剂化物或前药，其中，Z1是N。
- 根据权利要求1-10任一项的肽或其药学上可接受的盐、溶剂化物或前药，其中，Z1是P。
- 根据权利要求1-12任一项的肽或其药学上可接受的盐、溶剂化物或前药，其中，Z3是A。
- 根据权利要求1-13任一项的肽或其药学上可接受的盐、溶剂化物或前药，其中，X2是Q。
- 根据权利要求1-14任一项的肽或其药学上可接受的盐、溶剂化物或前药，其中，Z2是T。
- 根据权利要求1-13任一项的肽或其药学上可接受的盐、溶剂化物或前药，其中，X2是C，Z2是C，X2和Z2的氨基酸侧链形成二硫键共价连接。
- 根据权利要求1-13任一项的肽或其药学上可接受的盐、溶剂化物或前药，其中，X3是D,Z2是K，X3和Z2的氨基酸侧链形成内酰胺键共价连接。
- 根据权利要求1-17任一项的肽或其药学上可接受的盐、溶剂化物或前药，其中，R1为乙酰基。
- 根据权利要求1-18任一项的肽或其药学上可接受的盐、溶剂化物或前药，其中，R2为氨基。
- 根据权利要求1-19任一项的肽或其药学上可接受的盐、溶剂化物或前药，其中，所述肽由5至13个氨基酸组成，优选由7、8、9、10或11个氨基酸组成。
- 根据权利要求1-20任一项的肽或其药学上可接受的盐、溶剂化物或前药，其中，所述肽为线性肽或环肽。
- 根据权利要求1-21任一项的肽或其药学上可接受的盐、溶剂化物或前药，其中，所述肽为环肽，优选地，所述环肽为首尾成环、DK侧链成环、或二硫键成环，其中，所述肽的环化区域包含核心基序(DXED),优选地，所述肽通过第3位和第8位氨基酸的侧链偶联成环、或通过第2位和第8位氨基酸的侧链偶联成环，或优选地，其中，X3是D，Z2是K，且所述肽通过X3与Z2的侧链之间形成的内酰胺键环化；或优选地，其中，X2是C，Z2是C，且所述肽通过X2与Z2的侧链之间形成的二硫键环化，再优选地，当Z2是氨基酸C或K时，X7由Z1Z2组成，且X1为T或S。
- 根据权利要求1-22任一项的肽或其药学上可接受的盐、溶剂化物或前药，其中，所述肽是化学修饰的，例如，PEG修饰，脂质修饰，D型氨基酸替代、或非天然氨基酸替代。
- 选自SEQ ID NO：1至49之任一的肽或其药学上可接受的盐、溶剂化物或前药、或与所述肽在氨基酸序列上相差1个或2个氨基酸替代、缺失或添加的肽，优选地，所述替代、缺失和添加发生在核心基序(DXED)之外的氨基酸残基上，例如为保守氨基酸替代。
- 选自SEQ ID NO:6,22,25,26,33,40,43,44和46之任一的肽或其药学上可接受的盐、溶剂化物或前药。
- 制备根据权利要求1-25任一项所述的肽或其药学上可接受的盐、溶剂化物或前药的方法，优选地，所 述方法包括采用固相合成法合成所述肽。
- 一种药物组合物，其包含根据权利要求1-25任一项所述的肽或其药学上可接受的盐、溶剂化物或前药、以及药学上可接受载体。
- 一种预防或治疗TGF-β相关疾病的方法，包括向有需要的受试者施用预防或治疗有效量的根据权利要求1-25任一项所述的肽或其药学上可接受的盐、溶剂化物或前药、或根据权利要求27所述的药物组合物，任选地，所述方法进一步包含向所述受试者施用有效量的第二治疗剂；优选地，所述疾病具有病变部位的巨噬细胞浸润，或与组织损伤、炎症或纤维化相关。
- 根据权利要求28所述的方法，其中所述TGF-β相关疾病是纤维化病症，优选地选自：肺纤维化(例如，IPF)，肝纤维化，肾纤维化，骨髓纤维化、心肌纤维化、和/或皮肤纤维化。
- 根据权利要求28所述的方法，其中所述TGF-β相关疾病是实体瘤，优选地选自：肺癌、肝癌、乳腺癌、子宫癌、前列腺癌、胰腺癌、结肠癌、皮肤癌、中枢神经系统癌症、纤维肌瘤、纤维瘤、纤维腺瘤和纤维肉瘤。
- 根据权利要求1-25任一项所述的肽或其药学上可接受的盐、溶剂化物或前药的用途，用于在体外或体内：-阻断CD36与TSP-1的结合；-抑制TSP-1依赖性的TGF-β1活化；-降低纤维化组织中的活性TGF-β1量；-抑制TGF-β1介导的下游信号传导；-减少纤维化组织中胶原沉积；-抑制由TGF-β1介导的炎性和/或纤维化病变；-抑制由TGF-β1刺激的胞外基质相关基因表达；-抑制肿瘤细胞迁移；-预防或治疗TGF-β1相关疾病，尤其是纤维化或癌症；或用于制备在上述用途中使用的药物。
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