A peptide analogue, compositions thereof and uses thereof

Abstract

The application discloses a peptide analogue, a composition thereof and use thereof. The application provides a compound as shown in formula II or a pharmaceutically acceptable salt thereof. The fatty acid modification of the peptide analogue of the application has a synergistic effect with XTEN / PAS modification. The peptide analogue of the application has a large molecular weight and good drug efficacy.

Description

[0001] This application claims priority to Chinese patent application 2025101302721, filed on February 5, 2025. The entire contents of the aforementioned Chinese patent application are incorporated herein by reference. Technical Field

[0002] This invention relates to a peptide analog, its composition, and its use. Background Technology

[0003] Thrombopoietin, also known as megakaryocyte growth-derived factor, is a glycosylated protein secreted and expressed by hepatocytes and kidney cells. It consists of 332 amino acids and has a molecular weight of 60-70 kDa. It is a key regulator in platelet production. It works by interacting with the c-MPl receptor (c-MPl receptors are mainly expressed on the surface of certain hematopoietic cells, such as megakaryocytes, platelets, and CD34). + Thrombopoietin binds to megakaryocytes (both primary and progenitor cells), causing receptor dimerization and activating downstream signaling pathways such as JAK / STAT, promoting megakaryocyte proliferation and differentiation to produce platelets. Clinically, thrombopoietin can be used to treat immune thrombocytopenic purpura, chemotherapy-induced cancer, spinal dysplasia syndrome, chronic hepatitis, aplastic anemia, and thrombocytopenia caused by hepatitis C virus infection. In 1995, two full-length glycosylated recombinant human TPO (rhTPO) and truncated non-glycosylated PEG-modified recombinant human TPO (PEG-rhTPO) produced by mammalian cells entered clinical trials, but these trials had to be terminated due to cross-reactivity with endogenous TPO caused by the production of anti-TPO antibodies. In 2006, Shenyang 3SBio Pharmaceutical Co., Ltd. approved the marketing of full-length glycosylated rhTPO produced by CHO cell expression in China. It is administered via once-daily subcutaneous injection and is used clinically for thrombocytopenia induced by chemotherapy for solid tumors.

[0004] Clackson et al. discovered that only a few key groups at the interface between human growth hormone and its receptor are actually involved in the vast majority of the binding activity. The remaining numerous protein ligands merely display binding epitopes in the correct topological structure. This makes it possible to find small, active ligands. For this reason, Cwirla et al. used phage display technology to screen and obtain the short peptide AF12505, with the amino acid sequence IEGPTLRQWLAARA; its IC50... 50 =2nM, EC 50 =400nM, and named it TMP (TPO mimic peptide). After dimerization, its in vitro activity was significantly enhanced (EC). 50 =0.1nM). TMP is not homologous to TPO in sequence, so it will not produce anti-TPO antibodies. However, like endogenous TPO, it acts on the extracellular domain of the c-Mpl receptor.

[0005] Based on the TMP sequence, Amgen developed romilastine, which was approved by the FDA in August 2008 for the clinical treatment of immune thrombocytopenic purpura (ITP). It combines TMP and Fc fragments, extending the half-life via FcRn in the circulation pathway, allowing for once-weekly subcutaneous injection. Romilastine is expressed as inclusion bodies in E. coli, containing four TMP molecules with a molecular weight of 59 kDa and multiple disulfide bonds, resulting in low production yield and significant quality control challenges. Furthermore, its Fc fragment facilitates placental crossing, making it unsuitable for use during pregnancy or in women planning to conceive.

[0006] CN111432845A discloses a group of polyethylene glycol-modified TPO mimic peptide dimers with the following amino acid sequence: IEGPTLRQX1aaLAARX2aa (SEQ ID NO: 15), where X1aa is tryptophan (W) or β-(2-naphthyl)alanine (2-Nal), and X2aa is alanine (A) or sarcosine (Sar). The two TPO mimic peptides within the molecule are linked at their carboxyl ends by lysine amide to form a dimer, and methoxy polyethylene glycol (MPEG20000) is attached to each of the two N-termini.

[0007] CN116655770A discloses a group of fatty acid-modified TPO mimic peptide homotetramers, obtained through solid-phase synthesis or recombinant expression: NH2-Cys-Gly-Lys-Gly-Gly-Gly-Gly-Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala-COOH (SEQ ID NO: 16). The N-terminal cysteine ​​provides an α-amino group and / or Lys provides an ε-amino group for fatty acid modification. The tetramer formation is achieved through disulfide bonding between the -SH group of the N-terminal cysteine ​​residue.

[0008] CN105017408A discloses a group of polyethylene glycol-modified TPO mimic peptide homotetramers. These are NH2-Cys-Gly-Gly-Gly-Gly-Gly-Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala-COOH (SEQ ID NO: 17), obtained through solid-phase synthesis or recombinant expression. The tetramers form via a dual-activated polyethylene glycol molecule as a coupling agent, which, under certain conditions, forms a covalently coupled polyethylene glycol tetramer with the free thiol group of the N-terminal cysteine. The polyethylene glycol molecules have a strength between 20 kDa and 40 kDa.

[0009] This invention provides a peptide analog of Formula I or a pharmaceutically acceptable salt thereof.

[0010] In the formula, R0 is NH2-Ser-Pro- or NH2-; R1 is -(Gly)5-Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala-(Gly)8-Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala-COOH (SEQ IDNO: 12); R2 is a PAS or XTEN sequence; In Formula I, the carboxyl and amino groups at both ends are the carboxyl and amino groups on the amino acids they are attached to.

[0011] In some embodiments, the PAS sequence is a sequence consisting of Pro, Ala, and Ser, containing about 30 to about 400 amino acids; the PAS sequence contains about 50 to about 350, about 100 to about 300, about 150 to about 250 amino acids; about 30, about 50, about 100, about 150, about 200, about 250, about 300, about 350, about 400 amino acids, and its secondary structure is a random coil.

[0012] In some embodiments of the present invention, the PAS sequence has or consists of any of the following sequences:

[0013] The precise length of the XTEN used can be varied without affecting the activity of the TPO mimic peptide.

[0014] In some implementations, the XTEN sequence is selected from 4 to 6 amino acid sequences selected from Gly, Ala, Ser, Thr, Glu, and Pro, consisting of 20 to 3000 amino acids.

[0015] In some embodiments, the XTEN has a length of about 36, about 40, about 42, about 72, about 96, about 144, about 288, about 400, about 500, about 576, about 600, about 700, about 800, about 864, about 900, about 1000, about 1500, about 2000, about 2500, or up to about 3000 amino acid residues.

[0016] In some embodiments of the present invention, the XTEN sequence has or consists of any of the following sequences: .

[0017] In some embodiments, the peptide analog represented by Formula I is any of the following peptide analogs (where NH2 and COOH represent peptide ends): Peptide analog 1: NH2-Ser-Pro-Cys-PAS 100 -(Gly)5-Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala-Gly-Gly-Gly-Gly- Gly-Gly-Gly-Gly-Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala-COOH (SEQ ID NO: 5) Peptide analog 2: NH2-Cys-PAS 30 -(Gly)5-Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala-Gly-Gly-Gly-Gl y-Gly-Gly-Gly-Gly-Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala-COOH (SEQ ID NO: 6) Peptide analog 3: NH2-Cys-XTEN 144-(Gly)5-Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala-COOH (SEQ ID NO: 7) Peptide analog 4: NH2-Cys-XTEN 288 -(Gly)5-Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala-COOH (SEQ ID NO: 8) Peptide analog 5: NH2-Cys-(Gly)5-Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala-COOH (SEQ ID NO: 9) Peptide analog 6: NH2-Cys-XTEN 288 -(Gly)5-Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala-Lys-COOH (SEQ ID NO: 10) Peptide analog 7: NH2-Cys-XTEN 288-(Gly)5-Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala-Gly-Gly-Gly-Gly-Gly-G ly-Gly-Gly-Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala-(Gly)5-Lys-COOH (SEQ ID NO: 11).

[0018] In another aspect, the present invention provides a compound of formula III or a pharmaceutically acceptable salt thereof.

[0019] In the formula, n is an integer from 0 to 10; R2 is defined as described above; In the formula, Xaa is either absent or Lys; when Xaa is present, R4 is connected to the ε-NH2 of Lys; R0' is either Ser-Pro- or H; R1' is -(Gly)5-Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala-(Gly)8-Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala- (SEQ ID NO: 13) or -(Gly)5-Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala-(Gly)8-Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala- (SEQ ID NO: 14); R4 is either absent or an albumin-binding agent, with the general structural formula -(yaa). m1 -CO-(CH2) m2 -R 4a ; yaa can be 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA), D-alanine residue (D-Ala), β-alanine residue (β-Ala), 4-aminobutyric acid residue (GABA), 2-aminoisobutyric acid residue (Aib), 2-aminobutyric acid residue (Abu), arginine residue (Arg), aspartic acid residue (Asp), asparagine residue (Asn), cysteine ​​residue (Cys), D-glutamic acid residue (D-Glu), γ-glutamic acid residue (γ-Glu), glutamine residue (Gln), glycine residue (Gly), histidine residue (His), isoleucine residue (Ile), leucine residue (Leu), lysine residue (Lys), proline residue (Pro), phenylalanine residue (Phe), serine residue (Ser), tyrosine residue (Tyr), threonine residue (Thr), tryptophan residue (Trp), valine residue (Val), or methionine residue (Met). m1 is an integer from 0 to 10; m2 is an integer from 6 to 20; R 4a It is -CH3 or -COOH; R3 is a chemical bond or a multivalent linker connecting an albumin-binding agent, the structure of which is shown in Formula IV: the a and b ends are respectively connected to S; the general structural formula of R5 is -(zaa). m3 -CO-(CH2) m4 -R 5a ;

[0020] R 5a It is -CH3 or -COOH; m3 is an integer from 0 to 10; m4 is an integer from 6 to 20; Zaa can be 2-(2-(2-aminoethoxy)ethoxy)acetic acid residue (AEEA), D-alanine residue (D-Ala), β-alanine residue (β-Ala), 4-aminobutyric acid residue (GABA), 2-aminoisobutyric acid residue (Aib), 2-aminobutyric acid residue (Abu), arginine residue (Arg), aspartic acid residue (Asp), asparagine residue (Asn), cysteine ​​residue (Cys), D-glutamic acid residue (D-Glu), γ-glutamic acid residue (γ-Glu), glutamine residue (Gln), glycine residue (Gly), histidine residue (His), isoleucine residue (Ile), leucine residue (Leu), lysine residue (Lys), proline residue (Pro), phenylalanine residue (Phe), serine residue (Ser), tyrosine residue (Tyr), threonine residue (Thr), tryptophan residue (Trp), valine residue (Val), or methionine residue (Met).

[0021] In some implementations, n is 0 or 5.

[0022] In some implementations, m1 is 2-8, for example 6 or 3.

[0023] In some implementations, m2 is 14-18, for example, 16.

[0024] In some implementations, m3 is 3-6, for example, 4.

[0025] In some implementations, m4 is 14-18, for example, 16.

[0026] In some embodiments, the 2-(2-(2-aminoethoxy)ethoxy)acetic acid residue is Preferably, for Connect end a to end closest to R3.

[0027] In some implementations, the D-alanine residue is .

[0028] In some implementations, the β-alanine residue is .

[0029] In some implementations, the 4-aminobutyric acid residue is .

[0030] In some implementations, the 2-aminoisobutyric acid residue is .

[0031] In some implementations, the 2-aminobutyric acid residue is .

[0032] In some implementations, the arginine residue is or .

[0033] In some implementations, the aspartic acid residue is or .

[0034] In some implementations, the asparagine residues are or .

[0035] In some implementations, the cysteine ​​residue is .

[0036] In some implementations, the D-glutamic acid residue is or Preferred ; Preferred Connect end a to end closest to R3.

[0037] In some implementations, the γ-glutamic acid residue is or Preferred , Preferred Connect end a to end closest to R3.

[0038] In some implementations, the glutamine residue is or .

[0039] In some implementations, the glycine residue is .

[0040] In some implementations, the histidine residue is .

[0041] In some implementations, the isoleucine residue is .

[0042] In some implementations, the leucine residue is .

[0043] In some implementations, the lysine residue is , or Preferred ; Preferred Connect end a to end closest to R3.

[0044] In some implementations, the proline residue is .

[0045] In some implementations, the phenylalanine residue is .

[0046] In some implementations, the serine residue is .

[0047] In some implementations, the tyrosine residue is .

[0048] In some implementations, the threonine residue is .

[0049] In some implementations, the tryptophan residue is .

[0050] In some implementations, the valine residue is .

[0051] In some implementations, the methionine residue is .

[0052] In some embodiments, each zaa and yaa is independently a 2-(2-(2-aminoethoxy)ethoxy)acetic acid residue (AEEA, γ-glutamic acid residue, lysine residue, 4-aminobutyric acid residue, glutamic acid, serine residue, valine residue, or methionine residue).

[0053] In some implementations, each zaa and yaa is independent for , , , , , , , or .

[0054] In some implementations, each zaa and yaa is independently a 2-(2-(2-aminoethoxy)ethoxy)acetic acid residue, a γ-glutamic acid residue, or a lysine residue.

[0055] In some implementations, each zaa and yaa is independent for , or Or, each zaa and yaa is independent. , or .

[0056] In some implementations, (zaa)m3 is 2. 1 and 1 .

[0057] In some implementations, (zaa)m3 is 2. 1 and 1 .

[0058] In some implementations, (yaa)m1 is 2. and 4 Or 2 and 1

[0059] In some implementations, R3 has the following structure: .

[0060] In some implementations, R4 is selected from one of the following groups: Terminal connected to Xaa:

[0061] KM02 KM03.

[0062] In some implementations, n is 0, 5, or 10.

[0063] In some embodiments, the compound represented by Formula III is any of the following compounds: Compound 1:

[0064] Compound 2:

[0065] Compound 3:

[0066] Compound 4:

[0067] Compound 5:

[0068] Compound 6:

[0069] Compound 7:

[0070] Compound 8: Compound 9:

[0071] Compound 10:

[0072] Compound 11:

[0073] Compound 12:

[0074] Compound 13:

[0075] Compound 14: .

[0076] The present invention provides a pharmaceutical composition comprising a peptide analog of Formula I or a pharmaceutically acceptable salt thereof, or a compound of Formula II or a pharmaceutically acceptable salt thereof, wherein the pharmaceutical composition preferably further comprises a pharmaceutically acceptable carrier and / or excipients.

[0077] The present invention provides the use of an analogue of Formula I or a pharmaceutically acceptable salt thereof, or a compound of Formula II or a pharmaceutically acceptable salt thereof, or the above pharmaceutical composition in the treatment of thrombocytopenia.

[0078] The present invention provides the use of a peptide analog of Formula I or a pharmaceutically acceptable salt thereof, or a compound of Formula II or a pharmaceutically acceptable salt thereof, or the above pharmaceutical composition in the preparation of a medicament for treating thrombocytopenia.

[0079] The present invention provides a method for treating thrombocytopenia, comprising administering to a patient in need an effective amount of the peptide analog of Formula I or a pharmaceutically acceptable salt thereof, or the compound of Formula II or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition thereof.

[0080] The thrombocytopenia mentioned can be immune thrombocytopenia induced by clinical autoimmunity, thrombocytopenia caused by tumor chemotherapy, chronic hepatitis, etc., or thrombocytopenia caused by diseases such as aplastic anemia.

[0081] In some embodiments of the present invention, the drug or drug composition is administered by intravenous injection, subcutaneous injection, intraperitoneal injection or intramuscular injection, or orally in the form of tablets or liquid formulations.

[0082] The present invention provides a nucleic acid molecule for encoding a continuous Cys-R2-R1 sequence, wherein R2 and R1 are as previously described.

[0083] In some embodiments of the present invention, in order to better facilitate the expression of the above-mentioned coding sequence, a nucleotide sequence encoding an acid-sensitive or enzyme-sensitive tag protein may be linked to the 3′ end of the above-mentioned coding sequence.

[0084] In some embodiments of the present invention, the tag protein is a SUMO tag, which can be specifically cleaved by the Ulp1 enzyme.

[0085] The Cys-R1 sequence in this invention can also be obtained independently using solid-phase synthesis.

[0086] The present invention provides an expression vector comprising the nucleic acid molecules described above.

[0087] The present invention provides a host cell comprising the expression vector as described above or the nucleic acid molecule as described above, for example, the host cell having the nucleic acid molecule as described above integrated into its genome.

[0088] In some embodiments of the present invention, the host cell is *Escherichia coli* (E. coli). E. coli Methods for producing and expressing recombinant peptides in vitro and in prokaryotic host cells are known to those skilled in the art.

[0089] In this invention, the term "amino acid residue" refers to an amino acid that lacks a hydrogen atom on its amino group and a hydroxyl group on its carboxyl group, or both amino groups lack a hydrogen atom and both carboxyl groups lack a hydroxyl group. The amino and carboxyl groups can be attached to the same carbon atom or to different carbon atom.

[0090] In this invention, the term "pharmaceutically acceptable salt" refers to a salt prepared from a compound with a relatively non-toxic, pharmaceutically acceptable acid or base. When a compound contains a relatively acidic functional group, a base addition salt can be obtained by contacting the neutral form of such a compound with a sufficient amount of a pharmaceutically acceptable base in a pure solution or a suitable inert solvent. When a compound of this invention contains a relatively basic functional group, an acid addition salt can be obtained by contacting the neutral form of such a compound with a sufficient amount of a pharmaceutically acceptable acid in a pure solution or a suitable inert solvent. When a compound contains both relatively acidic and relatively basic functional groups, it can be converted into a base addition salt or an acid addition salt.

[0091] In this invention, the term "pharmaceutical excipients" refers to excipients and additives used in the production of pharmaceuticals and the preparation of prescriptions; they are substances included in pharmaceutical preparations other than the active ingredient. See the Pharmacopoeia of the People's Republic of China (2015 Edition), Part IV, or the Handbook of Pharmaceutical Excipients (Raymond C Rowe, 2009 Sixth Edition).

[0092] In this application, the term “treatment” means a therapeutic therapy. When a specific condition is involved, treatment means: (1) alleviating one or more biological manifestations of the disease or condition; (2) interfering with (a) one or more points in a biological cascade that causes or precipitates the condition or (b) one or more biological manifestations of the condition; (3) improving one or more symptoms, effects or side effects associated with the condition, or one or more symptoms, effects or side effects associated with the condition or its treatment; or (4) slowing the development of the disease or one or more biological manifestations of the condition.

[0093] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here.

[0094] Without violating common sense in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0095] The reagents and raw materials used in this invention are all commercially available.

[0096] The positive and progressive effects of this invention are as follows: This invention provides a novel modified TMP tetramer, which introduces an XTEN sequence through recombination and introduces an adipose side chain through chemical modification, thereby possessing a dual long-acting modification module. After a single injection in mice, it significantly promotes an increase in peripheral blood platelet count.

[0097] This invention designs a series of TMP tetramer analogs containing lipid acylation modules and / or PAS and / or XTEN modules. By comparing their platelet-raising effects in vivo, it demonstrates that, at the same molar dosage, fatty acid modification and XTEN modification have a synergistic effect in improving pharmacokinetics, outperforming single fatty acid-modified tetramers, outperforming single XTEN-modified tetramers, outperforming single PAS-modified tetramers, and outperforming PAS and fatty acid dual-modified tetramers. The larger the molecular weight, the better the platelet-raising effect. Attached Figure Description

[0098] Figure 1The graph shows the effect data of 0.51 nM / kg in Example 20.

[0099] Figure 2 The graph shows the effect data of 1.7 nM / kg in Example 20.

[0100] Figure 3 This represents the dose-effect relationship of compound 12 in Example 21.

[0101] Figure 4 The pharmacokinetic curves are shown in Example 22. Detailed Implementation

[0102] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0103] Example 1: Preparation of TPO mimic peptide analogs Cys-R2-R1, Cys-R2-R1-Lys, and Cys-R2-R1-(Gly)5-Lys

[0104] The TPO-mimicking peptide analogue can be any of the following sequences (NH2 and COOH in the sequence represent peptide ends): TPO mimetic peptide analog 1: NH2-Ser-Pro-Cys-PAS 100 -(Gly)5-Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala-Gly-Gly-Gly-Gly- Gly-Gly-Gly-Gly-Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala-COOH (SEQ ID NO: 5) TPO mimetic peptide analog 2: NH2-Cys-PAS 30 -(Gly)5-Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala-Gly-Gly-Gly-Gl y-Gly-Gly-Gly-Gly-Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala-COOH (SEQ ID NO: 6) TPO mimetic peptide analog 3: NH2-Cys-XTEN 144-(Gly)5-Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala-Gly-Gly-Gly-Gl y-Gly-Gly-Gly-Gly-Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala-COOH (SEQ ID NO: 7) TPO mimetic peptide analog 4: NH2-Cys-XTEN 288 -(Gly)5-Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala-Gly-Gly-Gly-Gl y-Gly-Gly-Gly-Gly-Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala-COOH (SEQ ID NO: 8) TPO mimetic peptide analog 5: NH2-Cys-(Gly)5-Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala-Gly-G ly-Gly-Gly-Gly-Gly-Gly-Gly-Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala-COOH (SEQ ID NO: 9) The Cys-R2-R1-Lys sequence is as follows: TPO mimetic peptide analog 6: NH2-Cys-XTEN 288 -(Gly)5-Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala-Gly-Gly-Gly-Gly- Gly-Gly-Gly-Gly-Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala-Lys-COOH (SEQ ID NO: 10) The Cys-R2-R1-(Gly)5-Lys sequence is as follows: TPO mimic peptide analog 7: NH2-Cys-XTEN 288-(Gly)5-Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala-Gly-Gly-Gly-Gly-Gly-G ly-Gly-Gly-Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala-(Gly)5-Lys-COOH (SEQ ID NO: 11) The above sequence was obtained through recombinant expression in *E. coli*. The preparation process is as follows: SUMO fusion tags and sequences were used for fusion expression. The fusion sequence was inserted into a plasmid and then transformed into competent cells. E. coli In BL21(DE3) bacteria, engineered strains for expressing the fusion protein were obtained through screening. These engineered strains underwent high-density fermentation, and the fusion protein was expressed in a soluble form in the cytoplasm.

[0105] When the sequence is SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:9, the following preparation process is adopted: The bacterial cells were resuspended in 20 mM PB, 5 mM EDTA, and 5 mM MTCEP at pH 7.2, with a solid content of 10%. After three high-pressure homogenizations, Ulp1 enzyme was added for digestion (1:30 v / v) at 32°C for 15 min. The precipitate was then adjusted to pH 3.0 with 6 M HCl, and the supernatant was collected by centrifugation. The supernatant was adjusted to pH 7.4 with sodium hydroxide and then subjected to QXL flow-through. Mobile phase A consisted of 25 mM Tris at pH 7.4, and mobile phase B consisted of 25 mM Tris, 1 Mtris, and pH 7.4. The flow-through was collected. The flow-through was adjusted to pH 3.0 and loaded onto Kromasil 300-10-C4 (21.2 mm × 250 mm) in a container. Mobile phase A consisted of 0.1% TFA / H2O (containing 2 mM MTCEP), and mobile phase B consisted of 0.1% TFA / ACN. The elution gradient was 23% B to 30% B, eluted for 14 CV. After collecting the target components, they were rotary evaporated and freeze-dried.

[0106] When the sequence is SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO: 11, the following preparation process is adopted: The bacterial cells were resuspended in 10 mM PB, 10 mM EDTA, and 2 mM TCCEP at pH 7.0, with a solid content of 10%. After three high-pressure homogenizations, Ulp1 enzyme (1:50 v / v) was added for digestion at 16°C for 15 min. The pH was then adjusted to 3.0 with 6 M HCl to precipitate the cells, and the supernatant was collected by centrifugation. The supernatant was adjusted to pH 4.5 with HCl and loaded into QXL packing material (Borglon). Mobile phase A consisted of 25 mM sodium acetate at pH 4.5, and mobile phase B consisted of 25 mM sodium acetate, 1 M NaCl, and pH 4.5. Elution was performed at 0–100% B for 10 CV, and the target fraction was collected. After adjusting the pH of the eluent to 2.0, it was loaded into a Sepax Bio C4 (300 Å, 10 μm, 10 mm × 250 mm) mobile phase. Mobile phase A was 0.1% TFA / H2O, and mobile phase B was 0.1% TFA / ACN. The elution gradient was 25% B to 35% B, eluting for 20 CV. The target fraction was collected and then rotary evaporated and lyophilized.

[0107] The molecular weight of the obtained samples was confirmed by LC-MS, and the results are shown in the table below:

[0108] Example 2: Synthesis of KM03 modified with side chain

[0109] A. Solid-phase synthesis of albumin binders

[0110] Weigh 1.00 g of 2-CTC resin and place it in the reactor of a peptide synthesizer. Add 10 mL of DCM and soak for 1 h. Weigh 2-3 times the amount of Fmoc-AEEA-OH and 4-6 times the amount of DIEA, dissolve them in 10 mL of DCM, and add them to the reactor for reaction. The reaction temperature is room temperature, and the reaction is carried out for 2 hours to complete the coupling of the first amino acid. Then wash the resin with DCM 6 times. Subsequently, add 10 mL of 20% PIP / DMF solution and mix for 30 min to remove the amino protecting group. Wash the resin with DCM 6 times to couple the second amino acid. Weigh three times the amount of Fmoc-AEEA-OH, HOBt, and DIC, dissolve them in 10 mL of DMF / DCM (1:1) mixed solvent, and carry out the reaction at room temperature. Monitor the reaction progress with ninhydrin; the reaction is complete when the reaction is colorless. Wash the resin with DCM 6 times. Then, continue the coupling reaction of γ-glutamic acid and octadecanoic acid according to the above coupling method. Repeat this cycle until all amino acids are coupled.

[0111] B. Cleavage and precipitation of acylated side chains

[0112] The lysis reagent was added at a ratio of 5 mL lysis reagent / 1 g resin, with a reagent ratio of TFE:DCM = 1:4 (V:V). The mixture was stirred at room temperature for 1 hour, filtered, and the solvent was removed by vacuum distillation at 40°C. 10 mL DCM was added to the vacuum distillation flask, and the solvent was removed by vacuum distillation again. This process was repeated 2-3 times. Finally, 3 mL DCM was added to dissolve the peptide, and 40 mL of ice-cold ether was added. The mixture was placed in a -20°C freezer for 20 min, centrifuged, and vacuum dried to obtain the albumin binding agent KM03.

[0113] ESI-MS molecular weight confirmation showed that M / Z = 830 [M+H]+, which is consistent with the theoretical molecular weight.

[0114] Example 3: Synthesis of KMO2 with modified side chain

[0115] The preparation process was as described in Example 2. The molecular weight was confirmed by ESI-MS, and M / Z = 1217.31 [M+H], which is consistent with the theoretical molecular weight.

[0116] Example 4: Synthesis of the multivalent linker KCM05

[0117] First, (2,5-dioxopyrrolidone-1-yl)-3,5-bis[(2-bromoacetyl)amino]benzoate was synthesized according to CN112839681A. Then, 18-[[(1S)-4-[2-[2-[2-[2-[2-[2-[[(1S)-5-amino-1-carboxy-pentyl]amino]-2-oxo-ethoxy]ethoxy]ethylamino]-2-oxo-ethoxy]ethoxy]ethylamino]-1-carboxy-4-oxo-butyl]amino]-18-oxo-octadecanoic acid was synthesized according to Example 1 and dissolved in 2% triethylamine. Dissolve (2,5-dioxopyrrolidone-1-yl)-3,5-bis[(2-bromoacetyl)amino]benzoate in ethanol and add it dropwise to 18-[[(1S)-4-[2-[2-[2-[2-[2-[2-[2-[[(1S)-5-amino-1-carboxy-pentyl]amino]-2-oxo-ethoxy]ethoxy]ethylamino]-2-oxo-ethoxy]ethoxy]ethylamino]-1-carboxy-4-oxo-butyl]amino]-18-oxo-octadecanoic acid at a molar ratio of 1:1.2. After reacting for 1-3 hours, add acetic acid to adjust the pH to 6.6, and rotary evaporate at 43°C until a solid precipitates. The white solid obtained by adding water is the desired product.

[0118] Purity: 40%; Theoretical molecular weight: 1235 Da; Actual molecular weight was verified to be correct by LC-MS.

[0119] Example 5: Synthesis of Compound 12

[0120] The compound was obtained by linking TPO mimic peptide 4 via the multivalent linker KCM05, and the specific preparation process is as follows: TPO mimic peptide 8 lyophilized powder was dissolved in 50 mM Tris, 10 mM EDTA, and 2 mM TCEP at pH 8.7 to a concentration of 20 mg / ml. At room temperature, 3 equivalents of KCM05 solution (dissolved in ethanol) were added while stirring. After reacting for 30 min, the pH was adjusted to 3.0 with acetic acid to terminate the reaction. The reaction mixture was loaded into a Sepax Bio C4 tank (10 μm, 300 Å, 10 × 250 mm), with mobile phase A being 0.1% TFA / H2O and mobile phase B being 0.1% TFA / ACN. The elution gradient was 30% B to 43% B, eluting for 26 CV. The target fraction was collected and then rotary evaporated and lyophilized.

[0121] The purity of compound 12 was 65.85%; the theoretical molecular weight was 61969.68 Da; and the actual molecular weight was 61968.8388 Da.

[0122] Implementation 6: Synthesis of Compound 11

[0123] The compound was obtained by linking TPO mimic peptide 3 (SEQ ID NO:7) via the multivalent linker KCM05, and was prepared according to the method in Example 5.

[0124] The purity of compound 11 was 62.45%; the theoretical molecular weight was 35543.48 Da; and the actual molecular weight was 35542.5408 Da.

[0125] Example 7: Synthesis of Compound 7

[0126] The compound was obtained by linking TPO mimic peptide 1 (SEQ ID NO:5) via the multivalent linker KCM05, and was prepared according to the method in Example 5.

[0127] The purity of compound 7 was 91.02%; the theoretical molecular weight was 25286.84 Da; and the actual molecular weight was 25286.2624 Da.

[0128] Example 8: Synthesis of Compound 8

[0129] The compound was obtained by linking TPO mimic peptide 2 (SEQ ID NO:6) via the multivalent linker KCM05, and was prepared according to the method in Example 5.

[0130] The purity of compound 8 was 90%; the theoretical molecular weight was 13898.38 Da; and the actual molecular weight was 13897.664 Da.

[0131] Example 9: Synthesis of Compound 14 The compound was obtained by linking TPO mimic peptide 5 (SEQ ID NO:9) via the multivalent linker KCM05, and was prepared according to the method in Example 5.

[0132] The purity of compound 14 was 93.07%; theoretical molecular weight: 9053.84 Da; actual molecular weight: 9054.72 Da.

[0133] Example 10: Synthesis of Compound 10

[0134] The compound was obtained by linking TPO mimic peptide 5 (SEQ ID NO:9) via the multivalent linker KCM05, and was prepared according to the method in Example 5.

[0135] The purity of compound 14 was 93.07%; theoretical molecular weight: 9053.84 Da; actual molecular weight: 9054.72 Da.

[0136]

[0137] The compound was obtained by linking TPO mimic peptide 4 (SEQ ID NO:8) via disulfide bonds, and the specific preparation process is as follows: SEQ ID NO:22 lyophilized powder was dissolved in 50 mM Tris at pH 8.0 to a concentration of 2 mM in 25 mL. Hydrogen peroxide (final concentration 0.1%) was added while stirring at room temperature. After reacting for 30 min, the pH was adjusted to 3 with 6 M HCl. The reaction solution was loaded into a Sepax BioC4 reactor (10 μm, 300 Å, 10 × 250 mm). Mobile phase A was 0.1% TFA / H2O (containing 2 mM TCCEP), and mobile phase B was 0.1% TFA / ACN. The elution gradient was 25% B to 35% B, eluting for 10 CV. The target fraction was collected, then rotary evaporated and lyophilized.

[0138] Compound 10 was obtained with a purity of 88%; the theoretical molecular weight was 60891.4 Da; and the actual molecular weight was 60889.7346 Da.

[0139] Example 11: Synthesis of Compound 11

[0140] The compound was obtained by linking TPO mimic peptide 3 (SEQ ID NO:7) via disulfide bonds and prepared according to the method in Example 10.

[0141] The purity of the TSM06 compound was 89.4%; the theoretical molecular weight was 34465.2 Da; and the actual molecular weight was 34467.114 Da.

[0142] Example 12: Synthesis of Compound 8

[0143] The compound was obtained by linking SEQ ID NO:20 via disulfide bonds and was prepared according to the method in Example 10.

[0144] The TSM05 compound was found to have a purity of 93%; a theoretical molecular weight of 12820.28 Da; and an actual molecular weight of 12820.4424 Da.

[0145] Example 13: Synthesis of Compound 5

[0146] The compound was obtained by linking TPO mimic peptide 1 (SEQ ID NO:5) via disulfide bonds and prepared according to the method in Example 10.

[0147] Compound 5 was obtained with a purity of 92.80%; its theoretical molecular weight was 24208.74 Da; and its actual molecular weight was 24208.3836 Da.

[0148] Example 14: Synthesis of Compound 13

[0149] The compound was obtained by linking TPO mimic peptide 5 (SEQ ID NO:9) via disulfide bonds and prepared according to the method in Example 10.

[0150] The purity of compound 13 was 95.74%; the theoretical molecular weight was 7976.61 Da; and the actual molecular weight was 7976.61 Da.

[0151] Example 15: Synthesis of Compound 1

[0152] The compound was first prepared by linking TPO mimic peptide 6 (SEQ ID NO: 10) with KCM05 to obtain an intermediate, which was then prepared according to the method in Example 5. The obtained intermediate was then modified with KM03 to obtain compound 1, and the specific preparation process is as follows: The lyophilized intermediate powder was dissolved in a 1% triethylamine solution to a final concentration of 1 mg / mL. KMO3 was dissolved in ethanol to a concentration of 2 mg / mL and slowly added dropwise to the peptide aqueous solution at a ratio of 1:3. The mixture was stirred at room temperature for approximately 1 hour. After the reaction was complete, 1M HCl solution was added to adjust the pH to 2-3, and acetonitrile was added to a final concentration of 10%. The solution was loaded onto a Sepax Bio C4 (10 μm, 300 Å, 10 × 250 mm) laminator for purification. Mobile phase A was 0.1% TFA / H2O, and mobile phase B was 0.1% TFA / ACN. The elution gradient was 30% B to 50% B elution for 40 CV. The target fraction was collected and then rotary evaporated and lyophilized.

[0153] The purity of compound 1 was 72.11%; the theoretical molecular weight was 63653.76 Da; the molecular weight was verified to be correct by LC-MS.

[0154] Example 16: Synthesis of Compound 2

[0155] The compound was obtained by first linking TPO mimic peptide 7 (SEQ ID NO:11) with KCM05 and then modifying it with KM03. It was prepared according to the method in Example 15.

[0156] The purity of compound 2 was 72.11%; the theoretical molecular weight was 64224.28 Da; and the molecular weight was verified to be correct by LC-MS.

[0157] Example 17: Synthesis of Compound 3

[0158] The compound was obtained by first linking TPO mimic peptide 6 (SEQ ID NO:10) with KCM05 and then modifying it with KM02. It was prepared according to the method in Example 15.

[0159] The purity of compound 3 was 72.11%; the theoretical molecular weight was 62186.96 Da; the molecular weight was verified to be correct by LC-MS.

[0160] Example 18: Synthesis of Compound 4

[0161] The compound was obtained by first linking TPO mimic peptide 7 (SEQ ID NO:11) with KCM05 and then modifying it with KM02. It was prepared according to the method in Example 15.

[0162] The purity of compound 4 was 72.11%; the theoretical molecular weight was 64998.68 Da; the molecular weight was verified to be correct by LC-MS.

[0163] Example 19: Bioactivity Detection

[0164] Referring to the article "Establishment and Validation of a Method for Detecting the Bioactivity of Recombinant Human Thrombopoietin Mimic Peptide-Fc Fusion Protein for Injection by MO7e Cell Proliferation Assay" (Journal of Pharmaceutical Analysis, 2018, 38(10): 1740-1747), the Mo7e cell count was determined using the Mo7e cell proliferation assay. The sample concentration and Mo7e cell proliferation curve were obtained and compared with the positive reference (Romilastine, self-made). The results are shown in the table below.

[0165] Example 20: In vivo efficacy detection

[0166] ICR mice, weighing 20-25g, were used, with 3 mice per group. The negative control group received PBS buffer, and the positive control group received romipexine solution (prepared in-house, preparation method according to patent CN113527508 A). The test groups consisted of compounds 14, 5, 6, 7, 9, 10, 11, and 12, administered at 0.51 nM / kg and 1.7 nM / kg. Blood samples were collected from the tail vein on days 0, 3, 4, 5, 6, 7, 8, 9, 10, and 12 post-administration to determine peripheral blood platelet counts. The efficacy data for 0.51 nM / kg are shown in Table 1 and [Table data would be inserted here]. Figure 1 As shown, the effect data for 1.7 nM / kg are presented in Table 2 and... Figure 2 As shown.

[0167] Table 1

[0168] Note: The dosage of compound 14 in Table 1 is 0.51 nM / kg.

[0169] Table 2

[0170] Note: The dosage of compound 14 in Table 2 is 22 nM / kg, and the dosage of the others is 1.7 nM / kg.

[0171] Experimental results showed that in the low-dose group, compounds 7, 11, 12, and 10 increased platelet counts in mice; in the high-dose group, all compounds increased platelet counts to varying degrees; among them, compound 12 and the self-made romistatin increased platelet counts by 3-4 times in both the low-dose and high-dose groups, with comparable levels. This invention demonstrates that fatty acid modification and XTEN / PAS modification have a synergistic effect, and that the larger the molecular weight, the better the efficacy.

[0172] Example 21: Dose-effect relationship investigation of compound 12

[0173] In normal ICR mice, compound 12 was administered via a single subcutaneous injection on day 1 at doses of 0.51 μM / kg, 1.69 μM / kg, and 5.1 μM / kg. The negative control group received PBS solution. Blood samples were then collected via tail vein before administration (day 0) and on days 3, 4, 5, 6, 7, 8, 9, 10, and 12 after administration for platelet count analysis. Results are as follows... Figure 3 As shown in the figure. The experimental results showed that the increase in peak platelet count in mice after injection of different doses of compound 12 was dose-related, and the time to reach the peak value was gradually delayed with increasing dose, similar to the dose-response relationship of roprostine.

[0174] Example 22: Pharmacokinetic studies of compounds 12, 10 and 14

[0175] SD rats (250–300 g) were randomly divided into groups of three, and each group received a single subcutaneous injection of compound 12, compound 10, and compound 14 at a dose of 1.69 nM / kg. Whole blood samples (200 µL) were collected from the orbital region at 0 h before administration and at 5 min, 0.5 h, 2 h, 4 h, 8 h, 12 h, 24 h, 48 h, 72 h, and 96 h after administration. The blood samples were added to EDTA-K2 coated tubes, centrifuged (3000 × g, 15 min, 4°C) to separate the plasma, and stored at -80°C.

[0176] Plasma samples were detected using a TPOR / anti-TMP antibody sandwich ELISA method. rhTPOR was coated onto 96-well high-adsorption ELISA plates, blocked with 5% skim milk-PBS solution, and plasma samples were added for capture. Finally, detection was performed using a combination of anti-TMP rabbit polyclonal antibody (Global Gene, P300645) and HRP-goat anti-rabbit IgG antibody (Shanghai Yamei, LF102). The concentration of the target compound (ng / mL) in each plasma sample was obtained, and pharmacokinetic parameters were calculated using a non-compartmental model. The pharmacokinetic parameters are shown in the table below:

[0177] Pharmacokinetic curves as follows Figure 4 As shown.

[0178] Experimental results showed that for TMP peptides, the XTEN module was more effective than the fatty acid module in prolonging the plasma half-life. Further introduction of the fatty acid module into compound 10 (XTEN modified) yielded compound 12, which, although its half-life was not prolonged, showed improved C... max and AUC (0→∞)The platelet count increased by approximately double, and correspondingly, the peak platelet count of compound 12 also increased. This demonstrates that XTEN modification and fatty acid modification have a synergistic effect.

Claims

1. A peptide analog of Formula I or a pharmaceutically acceptable salt thereof, ； In the formula, R0 is NH2-Ser-Pro- or NH2-; R1 is -(Gly)5-Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala-(Gly)8-Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala-COOH (SEQ IDNO: 12); R2 is a PAS or XTEN sequence; In Formula I, the carboxyl and amino groups at both ends are the carboxyl and amino groups on the amino acids they are attached to.

2. The peptide analog of Formula I as claimed in claim 1, characterized in that, The PAS sequence is a sequence composed of Pro, Ala and Ser, containing about 30 to about 400 amino acids, and its secondary structure is a random coil. And / or, the XTEN sequence is selected from 4 to 6 amino acid sequences selected from Gly, Ala, Ser, Thr, Glu, and Pro, consisting of 20 to 3000 amino acids; preferably, the XTEN has a length of about 36, about 40, about 42, about 72, about 96, about 144, about 288, about 400, about 500, about 576, about 600, about 700, about 800, about 864, about 900, about 1000, about 1500, about 2000, about 2500, or up to about 3000 amino acid residues.

3. The peptide analog of Formula I as described in claim 1, or a pharmaceutically acceptable salt thereof, characterized in that, The PAS sequence comprises about 50 to about 350, about 100 to about 300, or about 150 to about 250 amino acids; or, the PAS sequence comprises about 30, about 50, about 100, about 150, about 200, about 250, about 300, about 350, or about 400 amino acids. Preferably, the PAS sequence has or consists of any of the following sequences: And / or, the XTEN sequence has or consists of any of the following sequences: 。 4. The peptide analog of Formula I as described in claim 1, or a pharmaceutically acceptable salt thereof, characterized in that, The peptide analog represented by Formula I is any one of the following peptide analogs: Similar peptide 1:NH2-Ser-Pro-Cys-PAS 100 -(Gly)5-Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala-COOH(SEQ ID NO: 5); Similar peptide 2:NH2-Cys-PAS 30 -(Gly)5-Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala-COOH (SEQ ID NO: 6); Peptide analogue 3: NH2-Cys-XTEN 144 -(Gly)5-Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala-COOH (SEQ ID NO: 7); Peptide analogue 4: NH2-Cys-XTEN 288 -(Gly)5-Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala-COOH (SEQ ID NO: 8); Peptide Analog 5: NH2-Cys-(Gly)5-Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala-Gly-Gly-Gl y-Gly-Gly-Gly-Gly-Gly-Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala-COOH (SEQ ID NO: 9); Peptide analogue 6: NH2-Cys-XTEN 288 -(Gly)5-Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala-Lys-COOH (SEQ ID NO: 10); Peptide analogue 7: NH2-Cys-XTEN 288 -(Gly)5-Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala-(Gly)5-Lys-COOH (SEQ ID NO: 11).

5. A compound of formula III or a pharmaceutically acceptable salt thereof, ； In the formula, n is an integer from 0 to 10; R2 is defined as described in any one of claims 1-4; In the formula, Xaa is either absent or Lys; when Xaa is present, R4 is connected to the ε-NH2 of Lys; R0' is either Ser-Pro- or H; R1' is -(Gly)5-Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala-(Gly)8-Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala- (SEQ ID NO: 13) or -(Gly)5-Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala-(Gly)8-Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala- (SEQ ID NO: 14); R4 is either absent or an albumin-binding agent, with the general structural formula -(yaa). m1 -CO-(CH2) m2 -R 4a ; yaa can be 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA), D-alanine residue (D-Ala), β-alanine residue (β-Ala), 4-aminobutyric acid residue (GABA), 2-aminoisobutyric acid residue (Aib), 2-aminobutyric acid residue (Abu), arginine residue (Arg), aspartic acid residue (Asp), asparagine residue (Asn), cysteine ​​residue (Cys), D-glutamic acid residue (D-Glu), γ-glutamic acid residue (γ-Glu), glutamine residue (Gln), glycine residue (Gly), histidine residue (His), isoleucine residue (Ile), leucine residue (Leu), lysine residue (Lys), proline residue (Pro), phenylalanine residue (Phe), serine residue (Ser), tyrosine residue (Tyr), threonine residue (Thr), tryptophan residue (Trp), valine residue (Val), or methionine residue (Met). m1 is an integer from 0 to 10; m2 is an integer from 6 to 20; R 4a It is -CH3 or -COOH; R3 is a chemical bond or a multivalent linker connecting an albumin-binding agent, the structure of which is shown in Formula IV: the a and b ends are respectively connected to S; the general structural formula of R5 is -(zaa). m3 -CO-(CH2) m4 -R 5a ; ； R 5a It is -CH3 or -COOH; m3 is an integer from 0 to 10; m4 is an integer from 6 to 20; Zaa can be 2-(2-(2-aminoethoxy)ethoxy)acetic acid residue (AEEA), D-alanine residue (D-Ala), β-alanine residue (β-Ala), 4-aminobutyric acid residue (GABA), 2-aminoisobutyric acid residue (Aib), 2-aminobutyric acid residue (Abu), arginine residue (Arg), aspartic acid residue (Asp), asparagine residue (Asn), cysteine ​​residue (Cys), D-glutamic acid residue (D-Glu), γ-glutamic acid residue (γ-Glu), glutamine residue (Gln), glycine residue (Gly), histidine residue (His), isoleucine residue (Ile), leucine residue (Leu), lysine residue (Lys), proline residue (Pro), phenylalanine residue (Phe), serine residue (Ser), tyrosine residue (Tyr), threonine residue (Thr), tryptophan residue (Trp), valine residue (Val), or methionine residue (Met).

6. The compound of formula II as claimed in claim 5, or a pharmaceutically acceptable salt thereof, characterized in that, The compound represented by formula Ⅲ satisfies one or more of the following conditions. (1) n is 0 or 5; (1) m1 is 2-8, for example 6 or 3; (2) m2 is 14-18, for example 16; (3) m3 is 3-6, for example 4; (4) m4 is 14-18, for example 16; (5) The 2-(2-(2-aminoethoxy)ethoxy)acetic acid residue is Preferably, for End a is connected to the end closest to R3; (6) D-alanine residues are ; (7) β-alanine residues are ; (8) The 2-aminoisobutyric acid residue is ; (9) The 2-aminobutyric acid residue is ; (10) Arginine residues are or ; (11) The aspartic acid residue is or ; (12) Asparagine residues are or ; (13) Cysteine ​​residues are ; (14) D-glutamic acid residues are or Preferred ; Preferred End a is connected to the end closest to R3; (15) γ-glutamic acid residues are or Preferred , Preferred End a is connected to the end closest to R3; (16) Glutamine residues are or ; (17) Glycine residues are ; (18) Histidine residues are ; (19) The isoleucine residue is ; (20) Leucine residues are ; (21) Lysine residues are , or Preferred ; Preferred End a is connected to the end closest to R3; (22) The proline residue is ; (23) Phenylalanine residues are ; (24) Serine residues are ; (25) Tyrosine residues are ; (26) Threonine residues are ; (27) The tryptophan residue is ; (28) Valine residues are ; (29) Methionine residues are ; And (30) each zaa and yaa is independently a 2-(2-(2-aminoethoxy)ethoxy)acetic acid residue (AEEA, γ-glutamic acid residue, lysine residue, 4-aminobutyric acid residue, glutamic acid, serine residue, valine residue or methionine residue; preferably, each zaa and yaa is independently a 2-(2-(2-aminoethoxy)ethoxy)acetic acid residue (AEEA, γ-glutamic acid residue, lysine residue, 4-aminobutyric acid residue, glutamic acid, serine residue, valine residue or methionine residue); , , , , , , , or ; Preferably, each zaa and yaa is independently a 2-(2-(2-aminoethoxy)ethoxy)acetic acid residue, a γ-glutamic acid residue, or a lysine residue; more preferably, each zaa and yaa is independently a 2-(2-(2-aminoethoxy)ethoxy)acetic acid residue, a γ-glutamic acid residue, or a lysine residue. , or Alternatively, each zaa and yaa can be independent. , or ; More preferably, (zaa)m3 is 2. 1 and 1 or 2 1 and 1 ; and / or, (yaa)m1 is 2. and 4 Or 2 and 1 ; For example, R3 has the following structure: ； And / or, R4 is selected from one of the following groups, Terminal connected to Xaa: KM02 KM03.

7. The compound of formula II as claimed in claim 5, or a pharmaceutically acceptable salt thereof, characterized in that, The compound represented by Formula III is any one of the following compounds: Compound 1: ； Compound 2: ； Compound 3: ； Compound 4: ； Compound 5: ； Compound 6: ； Compound 7: ； Compound 8: ； Compound 9: ； Compound 10: ； Compound 11: ； Compound 12: ； Compound 13: ； Compound 14: 。 8. A pharmaceutical composition comprising a peptide analog of Formula I as claimed in any one of claims 1-4 or a pharmaceutically acceptable salt thereof, or a compound of Formula II as claimed in any one of claims 5-7 or a pharmaceutically acceptable salt thereof, wherein the pharmaceutical composition preferably further comprises a pharmaceutically acceptable carrier and / or excipient.

9. The use of a peptide analog of Formula I as described in any one of claims 1-4 or a pharmaceutically acceptable salt thereof, or a compound of Formula II as described in any one of claims 5-7 or a pharmaceutically acceptable salt thereof, or the use of the pharmaceutical composition as described in claim 8 in the preparation of a medicament for treating thrombocytopenia.

10. The use as described in claim 9, characterized in that, The thrombocytopenia mentioned above can be immune thrombocytopenia induced by clinical autoimmunity, thrombocytopenia caused by tumor chemotherapy or chronic hepatitis, or thrombocytopenia caused by aplastic anemia. And / or, the drug or drug composition may be administered by intravenous injection, subcutaneous injection, intraperitoneal injection or intramuscular injection, or orally in the form of tablets or liquid formulations.

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