CN112891512A

CN112891512A - Application of polypeptide compound in preventing or treating hepatic fibrosis

Info

Publication number: CN112891512A
Application number: CN202110087285.7A
Authority: CN
Inventors: 张斌智; 刘琦
Original assignee: Qingyuan Tuwei Anchuang Technology Development Co ltd
Current assignee: Qingyuan Tuwei Anchuang Technology Development Co ltd
Priority date: 2021-01-22
Filing date: 2021-01-22
Publication date: 2021-06-04
Anticipated expiration: 2041-01-22

Abstract

The invention discloses an application of a polypeptide compound in preparing a medicament for preventing or treating hepatic fibrosis, wherein the polypeptide compound contains an amino acid sequence as follows: Cys-Xaa2-Lys-Asn-Xaa5-Xaa6-Thr-Xaa8-Cys-COR₁(ii) a Wherein R is₁＝‑NH₂or-OH; xaa2 ═ Lys or Ser; xaa5 ═ Ser or Tyr; xaa6 ═ Pro or Lys; xaa8 ═ Leu or Ala. The polypeptide compound targets GPR55, and a large number of experimental studies prove that the polypeptide compound can obviously improve hepatic fibrosis and can be applied to preparation of medicaments for preventing or treating hepatic fibrosis.

Description

Application of polypeptide compound in preventing or treating hepatic fibrosis

Technical Field

The invention belongs to the technical field of biochemistry, and particularly relates to a new application of a polypeptide compound, namely an application of the polypeptide compound in treating or preventing hepatic fibrosis.

Background

Hepatic fibrosis is a progressive chronic liver disease that eventually progresses to cirrhosis (Don C, Rocky PDB, Joseph A. fibrosis-A common pathway to organ in liver failure [ J ]. New England and Journal of Medi-cine,2015,372(12):1138 and 1149). Various stimuli can cause chronic liver diseases such as toxins, viruses, cholestasis, hypoxia or harmful metabolites, etc. When stimulation is repeated or sustained over a long period of time, the injury increases collagen content in the extracellular matrix (ECM) and drives progressive fibrosis by inducing fibrogenic cytokines/growth factors and other mediators that activate downstream effectors of fibrosis. Collagen is the most abundant ECM component in fibrosis, increases by 10-fold in cirrhosis, and many ECM molecules can be used as both an index and a target for treatment of fibrosis. Wherein hepatic stellate cells, fibroblasts and sinus endothelial cells are stimulated to be activated in large amounts to produce harmful metabolites. With the progression of the disease process, in advanced fibrosis, the extracellular matrix is fibrotic by collagen (type I, type III and type V) secreted by activated hepatic stellate cells and fibroblasts, as well as other proteins constituting pathological fibrous tissues.

At present, the intervention drugs and therapies aiming at the etiology of hepatic fibrosis mainly comprise the following medicines: (1) specific inhibition of collagen synthesis therapy; (2) inhibition of collagen crosslinking therapy with lysyl oxidase and tissue transglutaminase; (3) small interfering RNA, antisense oligonucleotide and liver targeting nanocarrier therapy; (4) an integrin; (5) targeted Fibroblast Activity Protein (FAP) therapy. Although there are numerous drugs for treating hepatic fibrosis instead today, there are still no targeted drugs and therapies (Modern Practical Medicine, November 2020, vol.32, No.11 advances in anti-fibrosis and targeted drug therapy for hepatic fibrosis treatment, wangsi, chengili, fenpiceid).

GPR55 is a cannabinoid receptor expressed primarily in the pancreas, adipose tissue, gastrointestinal tract, liver and brain. Lysophosphatidylinositol is an endogenous ligand of GPR55, which binds to GPR55 and activates its downstream pathways, such as the RhoA-ROCK pathway and calcium ion release (Tuduri E, Imbernon M, Hernandez-Bautista RJ, Tojo M, Ferno J, Dieguez C, nogueras r.gpr55: a new formulating target for metablism J Mol Endocrinol 2017; 58: R191-R202). In recent years, it has been reported that the LPI/GPR55 system plays a crucial role in the development of nonalcoholic fatty liver diseases (Fondervia MF, FernandeZ U, Gonzalez-Rellan MJ, Da Silva Lima N, Buque X, Gonzalez-Rodriguez A, Alonso C, et al. the L-alpha-lysophosphatdylisinosite/GPR 55system indexes of the grade of non-alcoholic steatosides and steatopathies. hepatology 2020). Therefore, GPR55 may be a potential therapeutic target for alleviating chronic liver disease.

Disclosure of Invention

The invention aims to provide a new application of a polypeptide compound, namely an application of the polypeptide compound in preventing or treating hepatic fibrosis. The polypeptide compound targets GPR55, and a large number of experimental studies prove that the polypeptide compound can obviously improve hepatic fibrosis and can be applied to preparation of medicaments for preventing or treating hepatic fibrosis.

To achieve the above object, the present invention provides an application of a polypeptide compound in preparing a medicament for preventing or treating liver fibrosis, wherein the polypeptide compound comprises the following amino acid sequences:

Cys-Xaa2-Lys-Asn-Xaa5-Xaa6-Thr-Xaa8-Cys-COR₁

wherein R is₁＝-NH₂or-OH;

xaa2 ═ Lys or Ser;

xaa5 ═ Ser or Tyr;

xaa6 ═ Pro or Lys;

xaa8 ═ Leu or Ala.

The polypeptide compound of the present invention, preferably Xaa6 ═ Pro.

The polypeptide compound of the present invention is preferably Xaa2 ═ Lys.

The polypeptide compound of the present invention is any one of the following compounds 1 to 8:

compound 1(SEQ ID NO. 1):

Cys-Lys-Lys-Asn-Ser-Pro-Thr-Ala-Cys

CKKNSPTAC

compound 2(SEQ ID NO. 2):

Cys-Lys-Lys-Asn-Tyr-Pro-Thr-Leu-Cys

CKKNYPTLC

compound 3(SEQ ID NO. 3):

Cys-Lys-Lys-Asn-Tyr-Pro-Thr-Ala-Cys

CKKNYPTAC

compound 4(SEQ ID NO. 4):

Cys-Lys-Lys-Asn-Ser-Pro-Thr-Leu-Cys

CKKNSPTLC

compound 5(SEQ ID NO. 5):

Cys-Ser-Lys-Asn-Tyr-Pro-Thr-Leu-Cys

CSKNYPTLC

compound 6(SEQ ID NO. 6):

Cys-Ser-Lys-Asn-Ser-Pro-Thr-Leu-Cys

CSKNSPTLC

compound 7(SEQ ID NO. 7):

Cys-Ser-Lys-Asn-Ser-Pro-Thr-Ala-Cys

CSKNSPTAC

compound 8(SEQ ID NO. 8):

Cys-Ser-Lys-Asn-Tyr-Pro-Thr-Ala-Cys

CSKNYPTAC。

another aspect of the present invention is to provide a composition comprising the above polypeptide compound.

The composition is a pharmaceutical composition, and the polypeptide compound is used as an active ingredient and is added with a pharmaceutically acceptable carrier and/or an auxiliary material to prepare the pharmaceutical composition.

Cell and animal experiments show that the polypeptide compound of the invention has no adverse reaction and can be used for preventing or treating hepatic fibrosis.

It will be appreciated by those skilled in the art that the pharmaceutical compositions of the present invention are suitable for various modes of administration, such as oral, transdermal, intravenous, intramuscular, topical, nasal, and the like. Depending on the mode of administration employed, the pharmaceutical compositions of the polypeptide compounds of the present invention may be formulated into various suitable dosage forms comprising at least one effective dose of the polypeptide compound of the present invention and at least one pharmaceutically acceptable carrier. Examples of suitable dosage forms are tablets, capsules, sugar-coated tablets, granules, oral solutions and syrups, ointments and patches for the skin surface, aerosols, nasal sprays, and sterile solutions for injection.

Pharmaceutical compositions containing the polypeptide compounds of the present invention may be formulated as solutions or lyophilized powders for parenteral administration, which powders may be reconstituted with a suitable solvent or other pharmaceutically acceptable carrier prior to use, and liquid formulations are typically buffers, isotonic and aqueous solutions.

The amount of the pharmaceutical composition of the present invention may vary over a wide range and can be readily determined by one skilled in the art based on objective factors such as the type of disease, the severity of the condition, the weight of the patient, the dosage form, and the route of administration.

The invention has the advantages that:

1) the polypeptide compound of the invention has good biological activity;

2) the polypeptide compound can obviously improve hepatic fibrosis and has good treatment effect on the hepatic fibrosis;

3) the polypeptide compound of the invention shows significant pharmaceutical activity in the drug-induced experiments;

4) the polypeptide compound of the invention shows good safety and stability in the pharmaceutical experiment;

5) the polypeptide compound has high synthesis yield, good stability, easy scale-up production and low cost.

Abbreviations used in the present invention have the following specific meanings:

fmoc is fluorenylmethyloxycarbonyl, resin is resin, mPEG is monomethoxy polyethylene glycol, Cys is cysteine, Lys is lysine, Ser is serine, Asn is asparagine, Tyr is tyrosine, Thr is threonine, Ala is alanine, Leu is leucine, and Pro is proline.

Drawings

FIG. 1 shows the process at CCl₄In the induced hepatic fibrosis model of mouse liver, H of mouse liver after administration of 150. mu.g/kg of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5 and Compound 6, respectively&E staining pathological section picture.

FIG. 2 shows the result of the measurement in CCl₄Sirius red stained pathological section images of mouse livers after administration of 150 μ g/kg of compound 1, compound 2, compound 3, compound 4, compound 5 and compound 6, respectively, in an induced mouse liver fibrosis model.

FIG. 3 shows the result of the analysis of the signal at CCl₄Histogram analysis of liver lipid accumulation in mice following administration of 150 μ g/kg of compound 1, compound 2, compound 3, compound 4, compound 5 and compound 6, respectively, in the induced mouse liver fibrosis model.

FIG. 4 shows the result of the comparison between CCl₄Collagen1 immunohistochemical pathological section images of mouse liver after administration of 150 μ g/kg of compound 1, compound 2, compound 3, compound 4, compound 5, and compound 6, respectively, in the induced mouse liver fibrosis model.

FIG. 5 shows the result of the comparison between CCl₄Histogram analysis of liver lipid accumulation in mice following administration of 150 μ g/kg of compound 1, compound 2, compound 3, compound 4, compound 5 and compound 6, respectively, in the induced mouse liver fibrosis model.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

Example 1Synthesis of polypeptide Compounds

Materials:

all amino acids were purchased from gill biochemical (shanghai) ltd. All other reagents were analytically pure, Sigma brand, if not otherwise specified. Protein Technologies PRELUDE 6 channel polypeptide synthesizer was used. Phenomenex Luna C18 preparative columns (46 mm. times.250 mm) were used to purify the polypeptides. The high performance liquid chromatograph is a product of Waters company. Mass spectrometry was performed using an Agilent mass spectrometer.

The experimental method comprises the following steps:

the compound is synthesized from a carboxyl terminal to an amino terminal by adopting an Fmoc solid-phase polypeptide synthesis method and is sequentially connected according to the sequence of the amino acid sequence. The method comprises the following specific steps: firstly, MBHAR (Amide-MBHA-Resin Amide protected MBHA Resin) or Wang Resin is adopted to remove Fmoc protecting group for synthesis. And (3) detecting by using an indene detection reagent, wherein the use amount of the indene detection reagent is that the reagent 1: reagent 2: reagent 3 ═ l: 2: l (dropping), heating in boiling water for 3-10 min. Treating resin, weighing a certain amount of MBHA resin in a synthesizer, adding DCM, stirring for 30min, draining, washing with DMF for 4 times, each time for 2min, picking out a small amount of resin, performing indene detection, and if the resin color is not changed, indicating normal, using. Removing Fmoc protection: the 20% piperidine was washed 4 times, and after the 3 rd wash, it was indenylated, indicating that the next amino acid was accessible if the indenylated color appeared bluish-purple. Repeating the operation until the peptide synthesis is completed. Finally, washing the polypeptide after precipitation and cutting by utilizing ethyl acetate, and then purifying by high performance liquid chromatography.

Based on the above synthetic procedures, the following polypeptide compounds of the present invention were synthesized (table 1):

table 1. structure of the polypeptide compound synthesized in the examples of the present invention:

compound (I)	Numbering	Sequence of
			1	SEQ ID NO.1	Cys-Lys-Lys-Asn-Ser-Pro-Thr-Ala-Cys
2	SEQ ID NO.2	Cys-Lys-Lys-Asn-Tyr-Pro-Thr-Leu-Cys
			3	SEQ ID NO.3	Cys-Lys-Lys-Asn-Tyr-Pro-Thr-Ala-Cys
4	SEQ ID NO.4	Cys-Lys-Lys-Asn-Ser-Pro-Thr-Leu-Cys
			5	SEQ ID NO.5	Cys-Ser-Lys-Asn-Tyr-Pro-Thr-Leu-Cys
6	SEQ ID NO.6	Cys-Ser-Lys-Asn-Ser-Pro-Thr-Leu-Cys
			7	SEQ ID NO.7	Cys-Ser-Lys-Asn-Ser-Pro-Thr-Ala-Cys
8	SEQ ID NO.8	Cys-Ser-Lys-Asn-Tyr-Pro-Thr-Ala-Cys

Example 2The polypeptide Compound of the present invention para carbon tetrachloride (CCl)₄) Improvement and treatment effect of induced mouse hepatic fibrosis

In this embodiment, the polypeptide compound 1, the polypeptide compound 2, the polypeptide compound 3, the polypeptide compound 4, the polypeptide compound 5, and the polypeptide compound 6 are selected as examples to study the effect of the polypeptide compound of the present invention on improving and treating liver fibrosis in mice, and the specific method is as follows.

1. The tested drugs are: polypeptide compound analogs. The storage conditions were-20 ℃.

The molding method comprises the following steps: 48 male C57BL/6J mice, male, with a weight of 18-22g and a week age of 8 weeks (purchased from Beijing Wintonliflam laboratory animals technologies, Inc.), were randomly divided into 8 groups, each consisting of: 1) control group (Oil) + normal saline, i.e. intraperitoneal injection, n-6; 2) model control group (CCl)₄) Normal saline, intraperitoneal injection, n is 6; administration of CCl to mice₄Once in three days for 6 weeks; 3) model administration group (CCl)₄) +150 μ g/kg of polypeptide compound 1, i.p., n ═ 6/group; 4) model administration group (CCl)₄) +150 μ g/kg of polypeptide compound 2, i.p., n ═ 6/group; 5) model administration group (CCl)₄) +150 μ g/kg of polypeptide compound 3, i.p., n ═ 6/group; 6) model administration group (CCl)₄) +150 μ g/kg polypeptide compound 4, i.p., n ═ 6/group; 7) model administration group (CCl)₄) +150 μ g/kg of polypeptide compound 5, i.p., n ═ 6/group; 8) model administration group (CCl)₄) +150 μ g/kg polypeptide compound 6, i.p., n-6/group. Wherein mice are administered 20% CCl₄Control mice were given the same volume and frequency of corn oil injection at a dosing volume of 5.0 μ L/g of mouse body weight. The administration frequency is as follows: administration of CCl to mice₄Once in three days for 6 weeks; when the model administration group is given to mice CCl₄After 3 weeks, polypeptide compounds 1, 2, 3, 4, 5, 6 were administered for 3 weeks, respectively, starting at week 4. Wherein CCl₄And Oil was purchased from Shanghai Allantin Biotechnology Ltd.

2. And (3) evaluating the drug effect:

in CCl₄In the induced mouse hepatic fibrosis model, the mouse liver is characterized in that: there is inflammatory cell infiltration around the central venous area, edema and degeneration of liver cells, and large amount of collagen fiber deposition in the region of the junction and between the liver lobules. Three weeks after dosing, mice were treated and sampled, serological index testing was performed by retrobulbar venous bleeding, and liver tissue was taken for pathology analysis.

3. The experimental method comprises the following steps:

hematoxylin-eosin (H & E) staining: taking paraffin embedded tissue slices, and baking for 1h at 60 ℃. Dewaxing and hydrating: xylene 20 min → absolute ethanol 15 min → 95% ethanol 10min → 90% ethanol 5 min → 80% ethanol 5 min. Dyeing: hematoxylin for 7 minutes → tap water rinse clean → 1% hydrochloric acid ethanol differentiation 1s → tap water rinse → eosin staining 15s-20s → tap water rinse. And (3) dehydrating and transparency: 75% alcohol 1s → 85% alcohol 1s → 95% alcohol 1s → 100% alcohol 1s → xylene 1 s. The seal was air dried for 30 minutes and the gum was sealed.

Dyeing with sirius red: baking and dewaxing; standing in double distilled water for 5.0 minutes; dyeing with sirius red for 60-80 minutes in a dark room; rinsing with 0.5% glacial acetic acid for 5 s; dehydrating and transparentizing, sealing and taking a picture.

Immunohistochemistry: baking, dewaxing, and soaking in double distilled water for 5 min. Antigen retrieval: placing the rack for placing pathological sections in a buffer solution (PH is 6.0) beaker containing citric acid, and carrying out high temperature and high pressure for 15 minutes; the beaker was taken out, left to stand at room temperature until the temperature dropped to room temperature, then the sheet was taken out, put into 3.0% hydrogen peroxide for 10 minutes to block the endogenous peroxidase activity, and then washed 3 times with PBS for 5 minutes each. Tissues were blocked with 1.0% BSA for 1 h. Removing 1.0% BSA, adding Collagen I (type I Collagen) antibody on the tissue according to the recommended proportion of the instruction, and standing overnight at 4 ℃; taking out pathological sections the next day, adding a secondary antibody with horseradish peroxidase after the temperature is recovered, incubating for 60 minutes at 37 ℃, and then developing with DAB (diaminobenzidine) (TH & Ermo Scientific, USA); counterstaining with hematoxylin staining solution, washing with tap water for 5 minutes, differentiating with 1% hydrochloric acid ethanol, washing with tap water for 5 minutes, dehydrating, air-drying, sealing, and photographing (H & E staining solution and sirius red staining solution are from Shanghai Biotech engineering Co., Ltd., Collagen I antibody is from CST Co.).

In CCl₄In the induced mouse hepatic fibrosis model, the mouse liver is characterized in that: there is inflammatory cell infiltration around the central venous area, edema and degeneration of liver cells, and large amount of collagen fiber deposition in the region of the junction and between the liver lobules.

In CCl₄In the induced mouse liver fibrosis model, after 150 mug/kg of polypeptide compound 1, polypeptide compound 2, polypeptide compound 3, polypeptide compound 4, polypeptide compound 5 and polypeptide compound 6 are respectively administered, H of the mouse liver&The staining results are shown in FIG. 1; the results of sirius red staining and lipid accumulation analysis of mouse liver are shown in fig. 2 to 3; the immunohistochemical results of mouse livers are shown in FIGS. 4 to 5.

As can be seen from the results in FIG. 1, the aggregation of liver inflammatory cells of mice in each treatment model group is significantly improved after the mice with liver fibrosis are treated by the administration of the polypeptide compounds 1, 2, 3, 4, 5 and 6 of the present invention; liver fibrosis of mice in each model-administered group was significantly improved after administration of

polypeptide compounds

1, 2, 3, 4, 5 and 6. The polypeptide compounds 1, 2, 3, 4, 5 and 6 can well inhibit the accumulation of ECM, and treat and improve liver fibrosis.

As can be seen from the results of FIGS. 2 and 3, the hepatic fibrosis mice treated with the polypeptide compounds 1, 2, 3, 4, 5 and 6 of the present invention showed significant therapeutic improvement in hepatic collagen deposition in the mice of each treatment model group.

As can be seen from the results of FIGS. 4 and 5, the expression level of the liver Collagen I of the mice of each treatment model group is obviously improved after the mice with liver fibrosis are treated by the administration of the polypeptide compounds 1, 2, 3, 4, 5 and 6 of the invention.

In conclusion, the polypeptide compound can obviously improve hepatic fibrosis and has good treatment effect on hepatic fibrosis.

The present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof, and it is therefore intended that all such changes and modifications as fall within the true spirit and scope of the invention as defined by the appended claims be interpreted in accordance with the breadth to which they are fairly, if not explicitly recited herein.

Sequence listing

<110> Qing Yuan City chart microampere scientific and technological development Co., Ltd

Application of <120> polypeptide compound in preventing or treating hepatic fibrosis

<130> GD2390-21P125282

<160> 8

<170> PatentIn version 3.5

<210> 1

<211> 9

<212> PRT

<213> Artificial sequence

<400> 1

Cys Lys Lys Asn Ser Pro Thr Ala Cys

1 5

<210> 2

<211> 9

<212> PRT

<213> Artificial sequence

<400> 2

Cys Lys Lys Asn Tyr Pro Thr Leu Cys

1 5

<210> 3

<211> 9

<212> PRT

<213> Artificial sequence

<400> 3

Cys Lys Lys Asn Tyr Pro Thr Ala Cys

1 5

<210> 4

<211> 9

<212> PRT

<213> Artificial sequence

<400> 4

Cys Lys Lys Asn Ser Pro Thr Leu Cys

1 5

<210> 5

<211> 9

<212> PRT

<213> Artificial sequence

<400> 5

Cys Ser Lys Asn Tyr Pro Thr Leu Cys

1 5

<210> 6

<211> 9

<212> PRT

<213> Artificial sequence

<400> 6

Cys Ser Lys Asn Ser Pro Thr Leu Cys

1 5

<210> 7

<211> 9

<212> PRT

<213> Artificial sequence

<400> 7

Cys Ser Lys Asn Ser Pro Thr Ala Cys

1 5

<210> 8

<211> 9

<212> PRT

<213> Artificial sequence

<400> 8

Cys Ser Lys Asn Tyr Pro Thr Ala Cys

1 5

Claims

1. The application of a polypeptide compound in preparing a medicament for preventing or treating hepatic fibrosis, wherein the polypeptide compound contains an amino acid sequence shown as the following:

Cys-Xaa2-Lys-Asn-Xaa5-Xaa6-Thr-Xaa8-Cys-COR₁

wherein R is₁＝-NH₂or-OH;

xaa2 ═ Lys or Ser;

xaa5 ═ Ser or Tyr;

xaa6 ═ Pro or Lys;

xaa8 ═ Leu or Ala.

2. The use of claim 1 wherein Xaa6 is Pro.

3. The use according to claim 2 wherein Xaa2 ═ Lys.

4. The use according to claim 1, wherein the polypeptide compound is any one of the following compounds 1 to 8:

compound 1(SEQ ID NO. 1):

Cys-Lys-Lys-Asn-Ser-Pro-Thr-Ala-Cys

compound 2(SEQ ID NO. 2):

Cys-Lys-Lys-Asn-Tyr-Pro-Thr-Leu-Cys

compound 3(SEQ ID NO. 3):

Cys-Lys-Lys-Asn-Tyr-Pro-Thr-Ala-Cys

compound 4(SEQ ID NO. 4):

Cys-Lys-Lys-Asn-Ser-Pro-Thr-Leu-Cys

compound 5(SEQ ID NO. 5):

Cys-Ser-Lys-Asn-Tyr-Pro-Thr-Leu-Cys

compound 6(SEQ ID NO. 6):

Cys-Ser-Lys-Asn-Ser-Pro-Thr-Leu-Cys

compound 7(SEQ ID NO. 7):

Cys-Ser-Lys-Asn-Ser-Pro-Thr-Ala-Cys

compound 8(SEQ ID NO. 8):

Cys-Ser-Lys-Asn-Tyr-Pro-Thr-Ala-Cys。

5. a composition comprising the polypeptide compound of any one of claims 1 to 4.

6. The composition of claim 5, wherein the composition is a pharmaceutical composition, optionally further comprising a pharmaceutically acceptable carrier or adjuvant.