WO2014161118A1

WO2014161118A1 - Pentapeptide, polypeptide, cyclopeptide derivative, and compound and medicament and application thereof

Info

Publication number: WO2014161118A1
Application number: PCT/CN2013/001486
Authority: WO
Inventors: 王凤山; 廉倩倩; 王爱军; 程艳娜; 曹吉超
Original assignee: 山东大学
Priority date: 2013-04-03
Filing date: 2013-12-02
Publication date: 2014-10-09
Also published as: CN103159831B; CN103159831A

Abstract

The present invention provides a pentapeptide, a polypeptide using the pentapeptide as an active center, a cyclopeptide derivative, a compound, and an immunosuppressive medicament using the pentapeptide as an active center and an application thereof in the immunosuppressive medicament. The pentapeptide has an amino acid sequence Ala-Glu-Trp-Cys-Pro shown in SEQ ID NO. 1, and is from a low-molecular-weight calf thymus immunosuppressive extract.

Description

Pentapeptide, polypeptide, cyclic peptide derivative, compound, medicament and application thereof

The present invention relates to a pentapeptide, a polypeptide having the active center, a cyclic peptide derivative and a compound, and an immunosuppressive drug having the same as an active center, and use thereof for use in an immunosuppressive drug.

Background technique

The thymus is an important central immune organ in mammals. In addition to providing a cellular microenvironment required for T cell development, the thymus also produces a variety of peptide hormones that affect the differentiation and maturation of T cells, as well as regulate the function of mature T cells, and are important for the development of the body's immunological functions. significance. At present, there are relatively deep understandings and studies on extracts or factors that enhance the immunity of the thymus in the thymus, such as thymosin and thymopentin, but there are few reports on the factors of immunosuppression in the thymus.

In 1960, Bullough and Laurence isolated an epithelial cell division inhibitor from epithelial cells and proposed the concept of Chalone. Inhibin is a type of endogenous cell growth inhibitory factor. Since the 1970s, inhibitory substances that inhibit lymphocyte division have been extracted from bovine, porcine, and human embryonic thymus. Studies have shown that they have immunosuppressive activity and are not cytotoxic. Foreign studies on the thymus source of immunosuppressive substances mainly refer to the inhibitory substance. Because the inhibitory substances extracted from the thymus are unknown mixtures, the researchers studied their composition and properties. Houck et al. isolated immunosuppressants with molecular weights of 30kD~50kD, containing nucleic acids, which were heat sensitive. Later, they were hydrolyzed by ribonuclease and ultrafiltered to recover immunosuppressive components and speculated that the inhibitors were A positively charged glycopeptide having a molecular weight of about 5 kD. The Kiger et al. of France obtained a lymphocyte inhibitory factor by gel filtration and ion exchange chromatography, and it was speculated that it might be a thermostable, nucleotide-binding basic polypeptide. Allen et al. believe that the biologically active substance of lymphocyte inhibitory factor is spermine, which has a molecular weight of only 202D, and exhibits specific immunosuppressive activity by binding a tissue-specific substance into a compact spermamine complex in the extract. Rijke et al found that the lymphocyte inhibitory factor is a complex of spermine-protein binding. In the lymphocyte transformation inhibition test, spermine is actually inhibited, but it is found in the study of spermine as a control. Cell transformation. Patt et al. demonstrated that the lymphocyte transformation inhibitory factor is a hydrophobic, acidic, thiol-containing peptide having a molecular weight of 500D to 600D. Blazsek demonstrated that the inhibitor of lymphocyte transformation is a component of pI5.9. Heating, addition of protein denaturant (4mol/L deionized urea) or protease can destroy its activity, but it is digested by ribonuclease. No effect. Although early researchers studied the nature of thymus-derived immunosuppressants, their recent studies have not been reported, and no single compound from which a structure is well defined has been found.

Summary of the invention The object of the present invention is to provide a pentapeptide, a polypeptide having the active center, a cyclic peptide derivative and a compound, and an immunosuppressive drug having the same as an active center, and for immunizing it, in order to overcome the deficiencies of the above prior art. Inhibition of application in drugs.

In order to achieve the above object, the present invention adopts the following technical solutions:

A thymic immunosuppressive pentapeptide derived from LMW-TISE-c (low molecular weight bovine thymus immunosuppressive extract) which is an amino acid sequence as shown in SEQ ID NO: Ala-Glu -Trp-Cys-Pro.

A polypeptide obtained by adding one or more of an L-amino acid, a D-amino acid, a hydroxyamino acid, a methylated amino acid or a heterocyclic amino acid from the N-terminus or the C-terminus thereof with the pentapeptide as an active center.

The heterocyclic amino acid is tryptophan, histidine or valine.

The N-terminal and C-terminal of the pentapeptide are linked to form a cyclic peptide derivative.

The N-terminal of the pentapeptide is acetylated, formylated, alkylated or C-terminally aminoacylated or esterified, which has a greater bioactivity, half-life, stability, etc. than the pentapeptide. improve.

The guanidation is methylated, ethylated, propylated or butylated.

The esterification is carried out by esterification with methanol, ethanol, butanol or hexanol.

An immunosuppressive drug whose active ingredient comprises the above-mentioned pentapeptide, polypeptide, cyclic peptide derivative or compound for use in the preparation of an immunosuppressive drug.

Use of the above pentapeptide, polypeptide, cyclic peptide derivative or compound for the preparation of an immunosuppressive drug.

The pentapeptide of the present invention can be obtained by a known method in the prior art. The chemical synthesis can be carried out using an automatic peptide synthesizer, and the short peptide sequence can be deduced into a nucleotide sequence, which is then cloned into an expression vector for biosynthesis.

The present invention provides an immunosuppressive drug whose active ingredient comprises the pentapeptide, a polypeptide which is an active center thereof, a cyclic peptide derivative and a compound. The above immunosuppressive drug may further comprise one or more pharmaceutically acceptable carriers. The carrier includes conventional diluents, excipients, fillers, binders, wetting agents, disintegrating agents, absorption enhancers, surfactants, adsorption carriers, lubricants, etc. in the pharmaceutical field, and may also be added if necessary. Agents, sweeteners, etc. The immunosuppressive drug of the present invention can be prepared into various forms such as an injection, a powder injection, an inhalation, a tablet, a powder, a capsule, and an oral solution. The above various dosage forms of the drug can be prepared according to a conventional method in the pharmaceutical field.

The present invention also provides the use of the pentapeptide, a polypeptide having the active center, a cyclic peptide derivative and a compound for the preparation of an immunosuppressive drug.

In particular, the pentapeptide is useful in the preparation of a medicament for use in autoimmune diseases, organ transplantation, allergic diseases, and inflammatory diseases.

When the School of Pharmacy of Shandong Medical University (now Shandong University) conducted thymosin development research in the mid-1980s, The thymus immunosuppressive drugs prepared by the acidic extraction method are mainly immunosuppressive activities, and the thymus preparation prepared by the alkaline extraction method is mainly immunosuppressive activity. Depending on the preparation method and the reported thymus-derived immunosuppressive drugs, the applicant named the extract prepared by the alkaline extraction method as thymic immunosuppressive extract (TISE). Through the study on the biological activity and pharmacological effects of TISE and the similarity and similarity of TISE activity in different animal thymus sources, TISE has inhibitory effects on cellular immunity and humoral immunity, and also inhibits non-specific immune function, which can effectively inhibit skin transplantation. The rejection is also resistant to type I allergies and type III allergies, and TISE activity is not species specific. This suggests the possibility of applicant TISE as a novel immunosuppressive drug.

The beneficial effects of the invention are:

In order to reduce the possibility of heterogeneous TISE for allergic reactions in human body, TISE (CISE-c) derived from calf thymus was prepared into a low molecular weight TISE-c with small molecular weight and strong biological activity by using ultrafiltration technology. (Low Molecular Weight TISE-c, LMW-TISE-c). On this basis, the micrococci nuclease degradation LMW-TISE-c and RP-HPLC separation were combined with acid hydrolysis to finally purify a polypeptide consisting of 5 amino acid residues with a molecular weight of 604.95D. '-AEWCP-C' (Al _a -Glu-Trp-Cys-Pro). The present invention is the first to obtain structurally defined components from the thymus immunosuppressive component for the first time in many years. The pentapeptide is derived from TISE, and the present invention can be used to prepare an immunosuppressive drug based on previous studies on the activity of TISE.

DRAWINGS

Figure 1 shows the inhibitory effects of different drug concentrations on ConA-stimulated mouse spleen lymphocyte proliferation. ## indicates that p<0.01 compared with the blank group, there is a significant difference; ** indicates that _P <0.01, compared with the Con A group, has a significant difference.

Figure 2 shows the inhibitory effects of different drug concentrations on LPS-stimulated mouse spleen lymphocyte proliferation. ## indicates that p<0.01 is significantly different from the blank group; ** indicates p<0.01 compared with the Con A group, and there is a significant difference.

Figure 3 shows the toxicity of different concentrations of drugs on mouse spleen lymphocytes.

Figure 4 shows the effect of different drug concentrations on the viability of RAW264.7 cells. ** indicates that p < 0.01 compared with the blank group, there is a significant difference.

Figure 5 shows the effect of different concentrations of drugs on LPS-stimulated phagocytosis of RAW264.7 cells. # indicates that compared with the blank group, p<0.05, there is a difference, ## indicates that compared with the blank group, p<0.01, there is a significant difference; * indicates that compared with the model group, p<0.05, there is a difference, ** Indicates that p < 0.01 compared with the model group, there is a significant difference.

Figure 6 shows the lung histological effects (HE staining) of different concentrations of drugs in ovalbumin (OVA)-induced mouse asthma model; a. blank group (X 100 ), b. model group (X 100), c tectal Dexon (100), d. TIPP IO mg/kg (X 100 ), e. TIPP 50 mg/kg (Χ 100). Figure 7 shows the effect of different concentrations of drugs on IL-5 content in alveolar lavage fluid of OVA-induced mouse asthma model. # indicates that compared with the blank group, p<0.05, there is a difference, ## indicates that compared with the blank group, p<0.01, there is a significant difference; * indicates that compared with the model group, p<0.05, there is a difference, ** Indicates that p < 0.01 compared with the model group, there is a significant difference.

detailed description

The invention is further described in the following with reference to the accompanying drawings and embodiments.

A thymic immunosuppressive pentapeptide (TIPP) derived from LMW-TISE-c (low molecular weight bovine thymus immunosuppressive extract) which is an amino acid sequence as shown in SEQ ID NO:

Ala-Glu-Trp-Cys-Pro.

The heterocyclic amino acid is tryptophan, histidine or valine.

The compound obtained by acetylation, formylation or C-terminal aminoacylation or esterification of the pentapeptide has a greatly improved biological activity, half-life, stability and the like of the pentapeptide.

The guanidation is methylated, ethylated, propylated or butylated.

The esterification is carried out by esterification with methanol, ethanol, butanol, or hexanol.

Example 1 Inhibition of spleen lymphocyte proliferation in mice by different concentrations of TIPP

Six to eight weeks old male Balb/c mice were sacrificed by cervical dislocation and soaked in 75% alcohol for 5 min. The mice were removed and placed in a sterile dish with the left ventral side facing up. Cut a small opening in the middle of the left ventral side of the mouse, tear open the skin, expose the abdominal wall, and see a red strip-shaped spleen. The peritoneum was lifted on the lower side of the spleen, cut open, turned upside down, the spleen was exposed, the spleen was lifted with tweezers, the connective tissue under the spleen was separated by ophthalmology, and the spleen was taken out. Place in a Petri dish and rinse twice with Hank's solution 5 mL. Place the spleen on a stainless steel mesh (200 mesh), gently squeeze the spleen with a syringe plunger, rinse with 7-8 mL of Hank's solution, collect the cell suspension in a 10 mL sterile centrifuge tube, and centrifuge at 1000 r/min for 10 min. Discard the supernatant, add 8 mL of red blood cell lysate, mix well, let stand for 5 min, centrifuge at 1000 r/min for 10 min, discard the supernatant, and wash once with Hank's solution 8 mL. Finally, the cells were resuspended in 5 mL of RPMI-1640 medium (containing 10% fetal bovine serum), counted, and the cell concentration was adjusted to 2 χ 10 ⁶ /mL. Inoculate in a 96-well plate, 100 μΙ 7 wells, and set a zero hole. The cells were stimulated to grow with concanavalin Con Α (final concentration 5 g/mL) and lipopolysaccharide (LPS, final concentration l O g/mL). The groupings were: blank group, simple Con A or LPS stimulation group, cyclosporine Intravenous A (CsA) control group (Con A or LPS stimulation, CsA concentration 0.1 μιηοΙ / L), different concentrations of TIPP treatment group (Con A or LPS stimulation, TIPP final concentration of 50μηιο1/ί, 100 mol / L , 200μπΊθ1 ί, 300μΓηο1/ί, 400μηιο1/ί, 500 μηιοΙ/L), each set has 5 duplicate holes. After incubation at 37 ° C, 5% CO _{2 for} 68 h, add 5 mg / mL MTT 20 ^ L per well, continue to culture for 4 h at 37 ° C, 5% C0 ₂ , and centrifuge at 3000 r / min for 10 min. The supernatant was carefully aspirated with a 1 mL syringe, and 150 x L of DMSO was added to each well for 10 min. The OD value was measured at 570 nm by a microplate reader with a reference wavelength of 630 nm.

As shown in Fig. 1 and Fig. 2, Con A and LPS can cause significant proliferation of spleen lymphocytes; and CsA as a potent inhibitor of T cells can significantly inhibit the proliferation of spleen lymphocytes induced by Con A. The proliferation of splenic lymphocytes induced by LPS had no significant effect; TIPP inhibited the proliferation of spleen lymphocytes induced by Con A and LPS in a dose-dependent manner, and inhibited the proliferation of splenic lymphocytes induced by Con A. More obvious.

Example 2 Toxicity of different concentrations of drugs to mouse spleen lymphocytes

Six to eight weeks old male Balb/c mice were sacrificed by cervical dislocation and soaked in 75% alcohol for 5 min. The mice were removed and placed in a sterile dish with the left ventral side facing up. Cut a small opening in the middle of the left ventral side of the mouse, tear open the skin, expose the abdominal wall, and see a red strip-shaped spleen. The peritoneum was lifted on the lower side of the spleen, cut open, turned upside down, the spleen was exposed, the spleen was lifted with tweezers, the connective tissue under the spleen was separated by ophthalmology, and the spleen was taken out. Place in a Petri dish and rinse twice with Hank's solution 5 mL. Place the spleen on a stainless steel mesh (200 mesh), gently squeeze the spleen with a syringe plunger, rinse with 7-8 mL of Hank's solution, collect the cell suspension in a 10 mL sterile centrifuge tube, and centrifuge at 1000 r/min for 10 min. Discard the supernatant, add 8 mL of red blood cell lysate, mix well, let stand for 5 min, centrifuge at 1000 r/min for 10 min, discard the supernatant, and wash once with Hank's solution 8 mL. Finally, resuspend the cells in 5 mL of RPMI-1640 medium (containing 10% fetal bovine serum), count, adjust the cell concentration to lx lO ⁸ cells/mL, inoculate 96-well plates, 100 μυ^Ι, and set the zero-well . Different concentrations of cesium were added, and 5 replicate wells were set for each concentration. The concentrations are: 0, 0.0064 mol/L, 0.032 mol/L, 0.16 mol/L, 0.8 mol/L, 4 mol/L, 20 mol/L, 10 (^mol/L, 500 μηιοΙ/Lo 37 ° C, 5 % C0 ₂ cell culture incubator for 68 h. Add 5 mg/mL MTT 20 μί, continue to culture at 37 °C, 5% C0 _{2 for} 4 h, centrifuge at 3000 r/min for 10 min, and use 1 mL for supernatant. Syringe carefully aspirate, add DMSO 150 μί per well, shake for 10 min, and measure the OD value at 570 nm with a plate reader at 630 nm. Survival rate = (OD570nm in test group / OD570nm in normal group) χ 100%.

The results are shown in Fig. 3. After co-culture of spleen lymphocytes with TIPP for 68 hours, the survival rate was not significantly different from that of the TIPP group, that is, TIPP had no obvious cytotoxicity to spleen lymphocytes.

Example 3 Different concentrations of TIPP inhibited the proliferation of RAW264.7 cells

RAW264.7 cells in logarithmic growth phase cultured in DMEM medium (10% FBS) were adjusted to a cell density of 2χ 10 ⁴ /mL, seeded in 96-well plates at a volume of 100 μί per well, placed at 37 °C. , cultured for 2 h under 5% C0 ₂ conditions The cells were adherent, and different concentrations of TIPP were added (final concentrations of 25 g/mL, 50 g/mL, 100 g/mL, 200 g/mL, 400 g/mL, 5 replicates per concentration), Dexamethasone (Dex, final concentration of 5 g/mL) was used as a positive control. Continue to culture for 48 ho per well plus 5 mg/mL MTT 20 μL, continue culture at 37 °C, 5% C0 ₂ for 4 h, 3000 After r/m centrifugation for 10 min, the supernatant was carefully aspirated with a 1 mL syringe. Each well was incubated with DMSO 150 μί, oscillated for 10 min, and the OD value was measured at 570 nm with a microplate reader at 630 nm.

The result is shown in Figure 4. TIPP inhibited the proliferation of RAW264.7 cells at a concentration lower than 50 g/mL, while inhibited the proliferation of RAW264.7 cells at 100 g/mL and above. According to the cytotoxicity scale, when the relative cell proliferation rate is greater than 100% or 75% to 99%, the cytotoxicity is graded 0 or 1 and is considered to be cytotoxic, when the relative cell proliferation rate is 50. At %~74%, the cytotoxicity was grade 2 and mild toxicity. As can be seen from Fig. 4, when the TIPP concentration is 200 g/niL, the cell viability still reaches about 85%. It can be considered that TIPP is not cytotoxic to RAW264.7 at a concentration of 20 (^g/mL; but TIPP 40 (^) At g/mL, the cytotoxicity reached grade 2 with mild toxicity.

Example 4 Effect of different concentrations of drugs on LPS-stimulated phagocytosis of RAW264.7 cells

AW264.7 cells in logarithmic growth phase were inoculated on a % well plate, and cell suspension (2χ10 ⁵ /mL) was added to each well. 100 cells were cultured at 37 ° C, 5% C0 ₂ incubator overnight, and the supernatant was discarded. Replace with 2.5% FBS in DMEM medium, 100 μΙ 7 wells. Grouping, blank control group: DMEM medium supplemented with 2.5% FBS 100 μ model group: LPS was added to a final concentration of 1 g/mL; Dex group: Dex (final concentration was 5 g/mL) and LPS (final) The concentration was 1 g/mL); the administration group: TIPP (final concentrations were 25 g/mL, 5 (^g/mL, 10 (^g/mL, 20 (^g/mL, 400 μg/mL) and LPS (final concentration is 1 g/mL) 50 μί each, 5 sets of duplicate wells in each group. After incubation for 24 hours in 37 V, 5% C0 ₂ incubator, discard the supernatant and add 0.1% neutral red solution 100 Incubate at 37 °C, 5% C0 ₂ for 1 h, discard neutral red, wash 3 times with pre-warmed PBS, and deduct 100 times each time. Add acetic acid-ethanol (volume ratio 1: 1) Cell lysate 200 Ι7 wells were allowed to stand overnight at 4 ° C, and the OD value was measured at 490 nm.

The result is shown in Figure 5. Compared with the blank group, the OD value of the LPS group was significantly different from that of the blank group, indicating that LPS can enhance the phagocytosis of RAW264.7 cells. Compared with the LPS group, the Dex group and TIPP at 50, 100, 200, and 400 g/mL inhibited the LPS-stimulated RAW264.7 phagocytosis neutral red activity, and the OD value at the TIPP concentration of 100 g/mL The level of the blank group was comparable, indicating that the phagocytosis of macrophages induced by LPS was completely inhibited when the TIPP concentration was 100 g/mL.

Example 5 Effect of TIPP on OVA-induced mouse asthma model

SPF female Balb/(^J, rats, 5-6 weeks, were randomly divided into 6 groups, 12 rats in each group, respectively, blank group, model group, dexamethasone control group (3mg/kg), TIPP low dose group ( 2mg/kg), TIPP medium dose group (10mgkg), TIPP High-dose group (50 mg/kg in the same condition, model group and drug-administered group were intraperitoneally injected on day 1, 7, and 14 (ip) Alu-Gel-S containing 50 g OVA (sigma, Grade V) ( SERVA Electrophoresis GmbH 0.2mL, blank group ip normal saline. From the 28th day, the model group and the drug-administered group were challenged with 50 μL of 2 mg/mL OVA solution, and the blank group was intranasally dripped with saline. Group subcutaneous injection (ih) saline, model group ih saline, dexamethasone group ih dexamethasone (Dex, 3 mg/kg), TIPP low, medium and high dose groups ih ΤΙΡΡ 2 mg/kg, 10 mg /kg, 50 mg/kg. When the nose is dropped, the mice are anesthetized with ether to grasp the mice, and their bodies are unable to sag. 50 drops of 50% or 2 mg/mL OVA solution is used to make the solution breathe naturally with the mice. Inhalation. Repeated nasal challenge on the 29th, 30th, 31st, 32nd and 33rd day to establish a mouse asthma model. The samples were taken within 24-48 h after the last challenge. The mice were sacrificed by the neck, fixed in the supine position, 75% ethanol disinfected neck. Department of skin, open the thoracic tracheal intubation. Ligation of the left lung, right lung with cold PBS 0.4 mL for alveolar lavage, to ensure Uniform expansion, slowly injecting each time after slow injection to recover the lavage solution in 1.5 mL EP tube, repeat 2 times to ensure the recovery rate reaches 80%~90%. Centrifuge at 4 °C, 650x g for 5 min. The supernatant was dispensed, stored at -80 'C for cytokine detection. The left lung was taken, fixed with 4% formaldehyde solution, sectioned, and HE stained.

The results are shown in Figure 6 and Figure 7. The lung tissue sections of each group were stained with HE. Compared with the normal group, the model group had mucosal congestion and edema, thickened mucosa, and inflammatory cell infiltration. The lumen was filled with a lot of mucus and inflammatory cells infiltrated around the bronchial wall. . In the Dex and TIPP groups, inflammation was significantly reduced, and only a small amount of inflammatory cells infiltrated around the bronchial wall. It indicated that Dex and TIPP can inhibit airway inflammation in asthmatic mice and inhibit the infiltration of inflammatory cells in the airway.

Figure 7 shows the effect of lanthanum on IL-5 levels in alveolar lavage fluid of asthmatic mice. IL-5 is a Th2-type cytokine that is closely related to the development of asthma. It promotes eosinophil differentiation, survival and recruitment to the airways and assists B lymphocytes to produce antibodies. As can be seen from Figure 7, IL-5 levels were significantly elevated in the model group compared with the blank group, while both Dex and TIPP significantly reduced IL-5 levels in alveolar lavage fluid.

The above description of the specific embodiments of the present invention has been described with reference to the accompanying drawings, but is not intended to limit the scope of the present invention. Modifications or variations are still within the scope of the invention.

Claims

claims

1. A thymic immunosuppressive pentapeptide, characterized in that it has the amino acid sequence shown in SEQ ID No. 1: Ala-Glu-Trp-Cys-Pro.

2. A method using the pentapeptide of claim 1 as the active center, adding one or more of L-amino acids, D-amino acids, hydroxyl amino acids, methylated amino acids or heterocyclic amino acids from its N-terminus or C-terminus. The resulting peptide.

3. A cyclic peptide derivative obtained by connecting the N-terminus and C-terminus of the pentapeptide described in claims 1 to 2 to form a ring.

4. A compound obtained by acetylation, formylation, and benylation at the N-terminus of the pentapeptide described in claims 1 to 2, or aminoacylation and esterification at the C-terminus.

5. An immunosuppressive drug, characterized in that its active ingredients include the pentapeptide according to claim 1, the polypeptide according to claim 2, the cyclic peptide derivative according to claim 3 or the cyclic peptide derivative according to claim 4. compound.

6. Use of the pentapeptide of claim 1, the polypeptide of claim 2, the cyclic peptide derivative of claim 3 or the compound of claim 4 in the preparation of immunosuppressive drugs.