Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
  • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
  • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
  • C07K17/02—Peptides being immobilised on, or in, an organic carrier
  • C07K17/04—Peptides being immobilised on, or in, an organic carrier entrapped within the carrier, e.g. gel, hollow fibre
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
  • A61K38/10—Peptides having 12 to 20 amino acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
  • C07K17/02—Peptides being immobilised on, or in, an organic carrier
  • C07K17/08—Peptides being immobilised on, or in, an organic carrier the carrier being a synthetic polymer
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/04—Linear peptides containing only normal peptide links
  • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids

Definitions

• the invention relates to the field of medicine, in particular to an integrin blocker having tumor suppressor angiogenesis, integrin affinity and binding ability, the blocker is a polyethylene glycol modified polypeptide, and the integrin is blocked Broken polyethylene glycol modified polypeptides can be used for the treatment of solid tumors. Background technique
• tumor angiogenesis inhibitors 1 selective action on vascular endothelial cells, systemic toxic and side effects; 2 target cells are vascular endothelial cells, and drugs are easily accessible from the blood. 3 vascular endothelial cells with no or little mutation, not easy to produce drug resistance, can be used for a long time; 4 can be combined with radiotherapy and chemotherapy methods to reduce the toxic side effects of the latter.
• integrin blockers developed internationally have entered Phase II clinical trials. However, there are no similar or similar products entering the market in China, and it is very necessary to develop such drugs with independent intellectual property rights in China.
• ZL2005100403785 Highly effective inhibition of angiogenic polypeptides and preparation methods and applications thereof, and introduction of several integrin inhibitors, one of which is an integrin blocker polypeptide sequence: Ile-Val- Arg- Arg-Ala-Asp- Arg-Ala- Ala -Val-Pro-Gly-Gly-Gly-Gly-Arg-Gly-Asp, this sequence contains the integrin ligand sequence (Gly-Gly-Gly-Arg-Gly-Asp) and the neovascularization sequence (Ile) -Val-Arg-Arg-Ala-Asp-Arg-Ala-Ala-Val-Pro), wherein the integrin ligand sequence contains the RGD sequence (Arg-Gly-Asp), and the integr
• the polypeptide was confirmed to have a good antitumor effect by repeated in vitro and in vivo activity evaluation, and can significantly inhibit endothelial cell migration, inhibit tumor neovascularization, and thereby inhibit tumor growth.
• the half-life of the above polypeptide is short, and the administration of the intended clinical person is an intravenous infusion every day, which brings certain pain to the patient.
• the modification or modification of the molecular structure is a common method to solve the problem of short half-life and continuous drug administration.
• chemical modification is the most widely used.
• the commonly used chemical modifier is polyethylene terephthalate (PEG). ).
• PEG polyethylene terephthalate
• Glucan polyamino acid, polyanhydride, and the like.
• PEG is non-toxic, non-immunogenic, and water soluble. Point, approved by the US Food and Drug Administration (FDA) as an auxiliary raw material and modifier for pharmaceuticals.
• FDA US Food and Drug Administration
• the molecular weight increases and the glomerular filtration rate decreases.
• the barrier function of PEG protects the protein from hydrolysis by proteolytic enzymes and reduces the production of neutralizing antibodies, which contribute to protein.
• PEG modification may also affect the biological activity of the protein, and its effect is related to the modifier, the modification conditions and the nature of the protein itself.
• the optimal modification is determined by the preparation of PEG-modified proteins and biological activity studies. The study of PEG modification of synthetic small-molecule polypeptides started late, but has attracted the attention of many researchers. SUMMARY OF THE INVENTION
• the present invention has further studied mPEG-SC-Ile-Val-Arg-Arg-Ala-Asp-Arg-Ala-Ala-Val-Pro-Gly-Gly-Gly-Gly-Arg-Gly-Asp and found that it is It can treat a variety of tumors with reduced frequency of administration.
• a polyethylene glycol-modified integrin blocker HM-3 wherein the sequence of the integrin blocker is mPEG-SC-Ile-Val-Arg-Arg-Ala-Asp-Arg-Ala-Ala-Val- Pro-Gly-Gly-Gly-Gly-Arg-Gly-Asp, characterized in that the mPEG-SC has a molecular weight in the range of 500 to 20,000.
• a polyethylene glycol modified integrin blocker HM-3 for the preparation of a medicament for treating tumors, characterized in that the tumor originates from the stomach, skin, head and neck, thyroid, pancreas, Primary/secondary cancer or sarcoma of the lungs, esophagus, breast, kidney, gallbladder, colon/rectum, ovary, uterus, cervix, prostate, bladder, testis.
• beneficial effect esophagus, breast, kidney, gallbladder, colon/rectum, ovary, uterus, cervix, prostate, bladder, testis.
• the present invention is directed to the above-mentioned integrin blocker mPEG-SC 2Qk- HM-3 for treating various tumors, and has carried out a large number of in vitro and in vivo activity studies, and found that mPEG-SC 2Qk -HM-3 is reduced in the frequency of administration. It has maintained good activity in inhibiting the growth of various tumors, expanding its social and economic value. 2. The study found that the sequence Ile-Val-Arg-Arg-Ala-Asp-Arg-Ala-Ala-Val-Pro has the effect of inhibiting tumor angiogenesis.
• the arginine-glycine-aspartate (RGD) sequence is an important ligand for integrin.
• the Gly-Gly-Gly-Gly-Gly-Arg-Gly-Asp peptide containing the RGD sequence can also specifically recognize the integration.
• the integrin blocker polypeptide of the present invention has a pro-integrin family on the C-terminal junction of the sequence Ile-Val-Arg-Arg-Ala-Asp-Arg-Ala-Ala-Val-Pro having an inhibitory effect on angiogenesis
• the N-terminus of the integrin blocker polypeptide was specifically optimized for polyethylene glycol modification, and the final optimized sequence was: mPEG-SC 20 k-Ile-Val-Arg-Arg-Ala-Asp-Arg-Ala -Ala-Val-Pro-Gly-Gly-Gly-Gly-Arg-Gly-Asp, which contains PEG and a polypeptide of 18 amino acids.
• the RGD sequence in the molecule has integrin affinity and binding ability, and studies have shown that it works.
• the target is integrin aVW and Cd ⁇ binding, but the main binding target is still integrin aVW, and the sequence contains a neovascular inhibitory sequence, thereby inhibiting tumor neovascularization, thereby inhibiting tumor growth and metastasis.
• Polyethylene glycol (PEG) is a kind of macromolecular polymer with unique physicochemical properties. It has good biocompatibility, non-toxic and non-antigenic.
• protein and peptide drugs after PEG modification remain unchanged, and PEG modification can give protein and peptide drugs a variety of excellent properties: (1) increase stability, prolong plasma half-life; (2) reduce Immunogenicity and antigenicity; (3) reduce toxic side effects; (4) reduce the possibility of degradation by hydrolase, reduce the rate of removal by the kidney; (5) improve drug distribution and kinetic behavior.
• the target of the modified polyethylene glycol is unchanged, prolonging the in vivo half-life of the polypeptide molecule, reducing the clearance rate, reducing the immunogenicity and antigenicity, and the anti-tumor activity remains unchanged, but the expression is reduced.
• the frequency of administration, once modified, was once administered once a day, and once every 2-3 days.
• the inventors have learned through extensive experiments that the integrin blocker has a significant anti-tumor effect in vivo, and has less side effects, less dosage and lower cost.
• the polyethylene glycol modified integrin blocker polypeptide designed by the invention is scientific, reasonable, feasible and effective, and can be used as a therapeutic drug for treating human solid tumors, and provides new ideas and prospects for future drug development, and has significant Social value and market value.
• the half-life of the modified polypeptide HM-3 was 0.46 h, and the half-life after modification with mPEG-SC 2Qk was 20.13 h.
• Figure 1 Flow cytometry assay to detect binding of integrin blocker polypeptide to a target, where a is the first experiment - and b is the replicate experiment;
• Figure 3 Inhibitory effect of integrin blocker polypeptide on human esophageal cancer Ecl09 xenograft tumor in nude mice;
• Figure 4 Inhibitory effect of integrin blocker polypeptide on human nasopharyngeal carcinoma CE nude mouse xenograft tumor growth;
• Figure 5 Inhibitory effect of integrin blocker polypeptide on human thyroid carcinoma SW-579 xenograft xenograft tumor;
• Figure 6 Inhibitory effect of integrin blocker polypeptide on human gastric cancer MGC803 xenograft tumor in nude mice;
• Figure 7 Integrin resistance The inhibitory effect of the polypeptide on the growth of human pancreatic cancer SW-1990 xenograft tumor in nude mice;
• Figure 8 The inhibitory effect of integrin blocker polypeptide on the growth of human lung cancer H460 xenograft tumor in nude mice;
• FIG 9 Inhibitory effect of integrin blocker polypeptide on human breast cancer MDA-MB-231 xenograft tumor in nude mice;
• Figure 10 Integrin blocker polypeptide inhibits tumor growth of human gallbladder carcinoma GBC-SD xenograft in nude mice
• Figure 11 shows the inhibitory effect of integrin blocker polypeptide on human kidney cancer A498 xenograft xenograft tumor growth;
• Figure 12 Inhibitory effect of integrin blocker polypeptide on human colon cancer HT-29 xenograft xenograft tumor;
• Figure 13 Inhibition effect of integrin blocker polypeptide on human ovarian cancer SK-OV-3 xenograft tumor in nude mice;
• Figure 14 Integrin blocker polypeptide inhibits growth of human endometrial carcinoma HHUA nude mouse xenograft tumor
• Figure 15 shows the inhibitory effect of integrin blocker polypeptide on human cervical cancer HeLa nude mice
• Figure 17 Inhibitory effect of integrin blocker polypeptide on human prostate cancer DU-145 xenograft xenograft tumor;
• Figure 18 Inhibitory effect of integrin blocker polypeptide on human bladder cancer HT1376 xenograft tumor in nude mice;
• 19 in vivo map of integrin blocker polypeptide on human bladder cancer HT1376 xenograft xenograft tumor growth inhibition;
• Figure 20 Inhibitory effect of integrin blocker polypeptide on human testicular cancer 5637 xenograft tumor growth in nude mice;
• Figure 21 Inhibitory effect of integrin blocker polypeptide on sarcoma HT-1080 xenograft tumor growth in nude mice;
• the method comprises the steps of: using Fmoc-Ile-wang resin or Fmoc-lie-CTC resin as a starting material, and then sequentially contacting the dipeptide to the octapeptide with a protected amino acid, and washing the peptide sufficiently after the work is completed, and then cutting the peptide and then treating the peptide Get HM-3 crude.
• the crude product was purified, first dissolved, then purified twice with a preparative high performance liquid phase, and finally concentrated and lyophilized to give a pure product. Specific steps are as follows:
• Fmoc-lie-Wang resinl4.7g was weighed, poured into a 1 L glass sand core reaction column, and CH 2 C1 2 147 ml was added to fully expand the resin.
• Uncap the cap Add 25 ml of the dehydrogenating solution of the hexahydropyridine/DMF, seal and put in the shaker for 5 minutes. The temperature is controlled at room temperature. After 5 minutes, the capping solution is drained, washed once with DMF, and then added once again. 25 ml of the capping solution was reacted for 15 minutes;
• Post-treatment The polypeptide was precipitated by adding anhydrous ether to the cutting solution, then centrifuged, and the supernatant was poured off, and then the polypeptide was washed 6 times with anhydrous diethyl ether, and dried to obtain 9.5 g of crude polypeptide.
• Dissolution Weigh accurately the crude ID-18, add appropriate purified water to a solution of lOg/1, and ultrasonically stir to a granular clear solution.
• a solution that absorbs more than 200 mv at an ultraviolet wavelength of 220 nm is collected, and a purity of more than 95% is detected as a peak top, which is subjected to secondary separation and purification.
• Loading Evaporate the organic solvent from the peak received once, then apply the sample with an infusion pump at a flow rate of 80 ml/min, and collect the baseline to collect a solution that absorbs more than 200 mv at an ultraviolet wavelength of 220 nm to detect whether a sample has been washed out.
• a solution having a absorption of more than 200 mv at an ultraviolet wavelength of 220 nm was charged, and it was qualified by detecting a purity of more than 99%.
• Concentration, filtration, lyophilization The qualified solution was concentrated under reduced pressure at 37 ° C using a rotary evaporator to remove residual solvent and water for injection. Finally, it was filtered through a 0.22 um filter, and the filtrate was placed in a lyophilized tray, and freeze-dried by a freeze dryer to obtain a pure product.
• the prepared sample was purified by semi-preparative high performance liquid chromatography (HPLC, BIO-RAD) under the following conditions: mobile phase: ACN (+0.1% TFA), H 2 0 (+0.1% TFA); ACN linear gradient : 40%-95%; Flow rate: 2 ml/min; Running time: 12 min;
• Loading amount 1.0 ml; Detection wavelength: 220 nm.
• the product was collected using a centrifuge tube.
• the product collected by HPLC was first pre-freezed in a -70 ° C low temperature freezer overnight, and then lyophilized in a freeze-dried freeze dryer until all white powder was visually observed (about 30 h).
• the lyophilized product was harvested, the weight of the product was weighed and recorded, and stored in a refrigerator at -20 ° C and identified.
• the concentration of the concentrated gel was 5%, the concentration of the separating gel was 10%, the concentration voltage was 80 volts, and the separation voltage was 120 volts.
• the sample strip was first stained with Bal 2 .
• the PEG-containing fraction was stained; the marker was stained with Coomassie brilliant blue R250. After the dyeing was completed, it was placed in a decolorizing solution until the background was transparent, and then scanned for analysis.
• mPEG20000 modified integrin blocker polypeptide mPEG-SC 2 . k -HM-3
• the integrin blocker polypeptide referred to in the examples is an integrin blocker polypeptide modified by mPEG20000
• SD rats were randomly divided into 6 groups, male and female.
• Three groups of tail veins were given a high dose of integrin blocker peptide 52 mg/kg (equivalent to HM-3 4.2 mg/kg), a medium dose of 26 mg/kg (equivalent to HM-3 2.1 mg/kg), and a low dose of 13 Mg/kg (equivalent to HM-3 1.05 mg/kg), the other three groups were injected with HM-3 high dose 4.2 mg/kg, medium dose 2.1 mg/kg, Low dose of 1.05 mg/kg, rats in each group were collected from the orbital venous plexus at 0.5h, lh, 2h, 3h, 6h, 12h, 24h, 48h, 72h, 96h, 108h, 132h after administration.
• Table 1 mPEG-SC 2 . Comparison of pharmacokinetic parameters of k -HM-3 and HM-3 in SD rats. ⁇ /2 ⁇ is the half-life, which is the plasma clearance rate, and ⁇ f/C is the area under the curve, which is the average residence time.
• Tumor cells Bel-7402 and MCF-7 were cultured in a 24-well plate to 80% confluence, collected by trypsinization, washed twice with ice-cold PBS, and used with 1% BSA before labeling. Resuspend the PBS for 30 min.
• the cells were collected and washed twice with ice-cold PBS, then resuspended in 400 ⁇ M PBS, analyzed by flow cytometry, and FITC fluorescence was used to detect fluorescence intensity using the FL1 channel.
• mice were randomly divided into 2 groups, male and female, and 36 mg/kg of mPEG-SC 2 were administered to the tail vein. k- HM-3 and 3.0 mg/kg HM-3. After continuous administration for 8 weeks, blood was taken from the orbital venous plexus once a week for 1-12 weeks, centrifuged at 12000 rpm for 2 min, and the supernatant was separated and stored at -20 °C for storage. After being dissolved at room temperature, take 0.1 ml of supernatant Group settings:
• antibody production was observed at week 3 of the HM-3 group administration, while mPEG-SC 2 was used .
• Lower titers of antibodies were detected only at week 5 of the k- HM-3 group.
• the antibody titer of the integrin blocker polypeptide group was significantly lower than that of the HM-3 group, and the antibody titer decreased gradually after the drug was stopped. By the 12th week, the integrin blocker polypeptide group antibody could not be detected. This indicates that PEG modification can significantly reduce the immunogenicity of HM-3 in vivo.
• the relative tumor volume was calculated based on the measured results.
• the evaluation index of antitumor activity is the relative tumor growth rate T/C (%;), and the calculation formula is as follows:
• T/C (%) TRTV / CRT V X 100%
• the relative tumor volume was calculated based on the measured results.
• the evaluation index of antitumor activity is the relative tumor growth rate T/C (%;), and the calculation formula is as follows:
• T/C (%) TRTV / CRT V X 100%
• Dosing rate inhibition rate (mg/kg) weight (g) (g) Negative control once a day 22.78 12 22.37 12 1.201 a cisplatin 10 twice a week 22.35 8 22.33 7 0.320 73.36% Enrity 2.5 once a day 22.43 8 22.39 8 0.757 37.01%
• the inhibition rate of human nasopharyngeal carcinoma CE nude mice xenografts was 73.36%, but it had a significant effect on the body weight of experimental animals.
• 2.5mg/kg the tumor inhibition rate of human nasopharyngeal carcinoma CNE nude mice xenografts was 37.01%; HM-3 group 1.5mg/kg, the tumor inhibition rate of human nasopharyngeal carcinoma CE nude mice xenografts was 61.03
• the inhibition rate of high, medium and low dose groups of peptides in human nasopharyngeal carcinoma CNE nude mice was 68.22%, 66.19%, 55.32%, and had no significant effect on the weight of experimental animals.
• mPEG-SC 2 . k- HM-3 on human thyroid cancer SW-579 nude mouse xenograft tumor growth inhibition test The tumor tissue in the vigorous growth period was cut into 1.5mm 3 or so, under sterile conditions, inoculated into the right side of nude mice. .
• the transplanted tumors were measured with a vernier caliper, and the animals were randomly divided into groups after the tumors were grown to 100-200 mm 3 .
• the anti-tumor effect of the test polypeptide was dynamically observed using a method for measuring the tumor diameter. The number of measurements of the diameter of the tumor is once every 2 days, and the weight of the mouse is also weighed for each measurement. In the experimental group, the peptide was injected into the tail vein once every three days, and the negative group was given the same amount of normal saline at the same time.
• T/C (%) TRTV / CRT V X 100%
• the tumor inhibition rate was 32.20%; the HM-3 group 1.5mg/kg, the tumor inhibition rate of human thyroid cancer CNE nude mice xenografts was 57.70%; the polypeptide high, medium and low dose groups for human thyroid cancer SW-
• the tumor inhibition rate of 579 nude mice xenografts was 67.63%, 60.56%, 58.42%, and had no significant effect on the weight of nude mice.
• mPEG-SC 2 The growth inhibition test of k- HM-3 on human thyroid carcinoma SW-579 nude mice xenografts showed that compared with the negative control group, the 36.7 mg/kg peptide group had significant growth in human thyroid cancer SW-579 xenografts.
• Example 10 mPEG-SC 20k- HM-3 on human gastric cancer MGC803 nude mice xenograft tumor growth inhibition test About 1.5 mm 3 , under sterile conditions, inoculated into the right side of nude mice. The transplanted tumors were measured with a vernier caliper, and the animals were randomly divided into groups after the tumors were grown to 100-200 mm 3 . Use The method of measuring the tumor diameter dynamically observes the anti-tumor effect of the test polypeptide.
• the number of measurements of the diameter of the tumor is once every 2 days, and the weight of the mouse is also weighed for each measurement.
• the peptide was injected into the tail vein once every three days, and the negative group was given the same amount of normal saline at the same time.
• the relative tumor volume was calculated based on the measured results.
• the evaluation index of antitumor activity is the relative tumor growth rate T/C (%;), and the calculation formula is as follows:
• T/C (%) TRTV / CRT V X 100%
• paclitaxel group 10mg/kg the tumor inhibition rate of human gastric cancer MGC803 nude mice xenografts was 74.12%; Endo group 2.5mg/kg, the tumor inhibition rate of human gastric cancer MGC803 nude mice xenografts was 30.29%; HM-3 group 1.5mg/kg, the tumor inhibition rate of human gastric cancer MGC803 nude mice xenografts was 70.40%; The tumor inhibition rate of human gastric cancer MGC803 nude mice was 73.42%, 69.86%, 59.57%.
• the transplanted tumors were measured with a vernier caliper, and the animals were randomly divided into groups after the tumors were grown to 100-200 mm 3 .
• the anti-tumor effect of the test polypeptide was dynamically observed using a method for measuring the tumor diameter.
• the number of measurements of the diameter of the tumor is once every 2 days, and the weight of the mouse is also weighed for each measurement.
• the peptide was injected into the tail vein once every three days, and the negative group was given the same amount of normal saline at the same time.
• the relative tumor volume was calculated based on the measured results.
• the evaluation index of antitumor activity is the relative tumor growth rate T/C (%;), and the calculation formula is as follows:
• T/C (%) TRTV / CRT V X 100%
• mPEG-SC 20k- HM-3 on human lung cancer H460 nude mice xenograft tumor growth inhibition test The growth of the tumor tissue in the vigorous stage was cut into 1.5mm 3 or so, under sterile conditions, inoculated into the right side of nude mice .
• the transplanted tumors were measured with a vernier caliper, and the animals were randomly divided into groups after the tumors were grown to 100-200 mm 3 .
• the anti-tumor effect of the test polypeptide was dynamically observed using a method for measuring the tumor diameter. The number of measurements of the diameter of the tumor is once every 2 days, and the weight of the mouse is also weighed for each measurement. In the experimental group, the peptide was injected into the tail vein once every three days, and the negative group was given the same amount of normal saline at the same time.
• the relative tumor volume was calculated based on the measured results.
• the evaluation index of antitumor activity is the relative tumor growth rate T/C (%;), and the calculation formula is as follows:
• T/C (%) TRTV / CRT V X 100%
• Paclitaxel 10 once every two days 22.34 8 19.58 8 0.248 68.77% Enrity 2.5 once a day 21.08 8 20.86 8 0.546 31.20% HM-3 1.5 once every two days 21.33 8 21.11 8 0.274 65.42% mPEG-SC
• the paclitaxel group 10mg/kg, the tumor inhibition rate of human lung cancer H460 nude mice xenografts is 68.77%; Group 2.5mg/kg, the tumor inhibition rate of human lung cancer H460 nude mice xenografts was 31.20%; HM-3 group 1.5mg/kg, the tumor inhibition rate of human lung cancer H460 nude mice xenografts was 65.42%; The inhibition rate of high, medium and low dose groups on human lung cancer H460 nude mice xenografts was 66.45%, 55.37%, 54.28%.
• Example 13 mPEG-SC 20k- HM-3 on human breast cancer MDA-MB-231 xenograft tumor growth inhibition test in nude mice.
• the tumor tissue in the vigorous growth period was cut into 1.5 mm 3 or so under sterile conditions. Inoculated in the right side of the nude mice.
• the transplanted tumors were measured with a vernier caliper, and the animals were randomly divided into groups after the tumors were grown to 100-200 mm 3 .
• the anti-tumor effect of the test polypeptide was dynamically observed using a method for measuring the tumor diameter. The number of measurements of the diameter of the tumor is once every 2 days, and the weight of the mouse is also weighed for each measurement. In the experimental group, the peptide was injected into the tail vein once a day, and the negative group was given the same amount of normal saline at the same time.
• the relative tumor volume was calculated based on the measured results.
• the evaluation index of antitumor activity is the relative tumor growth rate T/C (%;), and the calculation formula is as follows:
• T/C (%) TRTV / CRT V X 100%
• RTV treatment group RTV
• CRTV negative control group
• RTV Table 8 mPEG-SC 20k -HM-3 inhibition of human breast cancer MDA-MB-231 xenograft tumor growth in nude mice Tumor inhibition rate Negative pair
• Paclitaxel 10 two days 23.90 8 23.32 0.335 73.05% Enrity 2.5 Daily times 24.65 8 23.03 0.801 35.57%
• Example 14 mPEG-SC 20k- HM-3 on human gallbladder carcinoma GBC-SD nude mice xenograft tumor growth inhibition test
• Human gallbladder carcinoma GBC-SD cell line in logarithmic growth phase was prepared under aseptic conditions 5 xl 0 7 /ml cell suspension was inoculated subcutaneously into the right axilla of nude mice with 0.1 ml.
• the nude mice xenografts were measured for the diameter of the transplanted tumor using a vernier caliper, and the animals were randomly divided into groups after the tumors were grown to 100-200 mm 3 .
• the effect of the anti-tumor effect of the test substance was dynamically observed using a method of measuring the tumor diameter.
• the number of measurements of tumor diameter was measured once every 2 days.
• the administration methods were all injected by tail vein.
• the negative control group was injected with the same amount of normal saline once a day; the paclitaxel group was 10 mg/kg once a week; the Endo group was 2.5 mg/kg, and the daily dose was 1 6, 3, 1.5 mg/kg, once a day. After the end of the administration, the mice were sacrificed, and the operation was enjoyed.
• Example 15 mPEG-SC 2 . k -HM-3 on A498 human renal carcinoma nude mouse xenograft tumor growth inhibition assay in logarithmic growth phase of human renal carcinoma cell line A498, the prepared 5 xl 0 7 / ml cell suspension in sterile conditions, The mice were inoculated subcutaneously in the right axilla of the nude mice with 0.1 ml. Nude mouse transplanted tumors with vernier calipers to measure the diameter of the transplanted tumor, to be tumor growth Animals were randomized after 100-200 mm 3 . The method of measuring the tumor diameter is used to dynamically observe the antitumor effect of the test substance. The number of measurements of tumor diameter was measured once every 2 days.
• the administration methods were all injected by tail vein.
• the negative control group was injected with the same amount of normal saline once a day; the paclitaxel group was 10 mg/kg once a week; the Endo group was 2.5 mg/kg, once a day; the high, medium and low peptides were 6, 3, respectively. 1.5 mg/kg, once a day.
• mice were sacrificed and the tumor pieces were surgically removed and weighed. Table 10. Inhibition of mPEG-SC 20k- HM-3 on human kidney cancer A498 xenograft tumor growth in nude mice
• Paclitaxel 10 two days 21.23 6 21. 15 0.242 74.32% grace 2.5 daily 21.43 6 21.66 0.656 30.51%
• Example 16 Inhibition of mPEG-SC 20k- HM-3 on xenograft tumor growth in human colon cancer HT-29 nude mice
• the human colon cancer HT-29 cell line in logarithmic growth phase was prepared as a 5 ⁇ 10 7 /ml cell suspension under sterile conditions, and inoculated into the right axilla of nude mice in 0.1 ml.
• the nude mice xenografts were measured for the diameter of the transplanted tumor using a vernier caliper, and the animals were randomly divided into groups after the tumors were grown to 100-200 mm 3 .
• the effect of the anti-tumor effect of the test substance was dynamically observed using a method of measuring the tumor diameter.
• the number of measurements of tumor diameter was measured once every 2 days.
• the administration methods were all injected by tail vein.
• the negative control group was injected with the same amount of normal saline once a day; the paclitaxel group was 10 mg/kg once a week; the Endo group was 2.5 mg/kg, once a day; the high, medium and low peptides were 6, 3, respectively. 1.5 mg/kg, once a day.
• the mice were sacrificed and the tumor pieces were surgically removed and weighed.
• Negative control one daily - - time 23.94 12 23.67 12 1.120 one paclitaxel 10 two days one - time 23.43 8 22.84 6 0.346 69.11% grace 2.5 daily - - time 23.23 8 22.81 8 0.745 33.48%
• HM-3 low results See Table 11 and Figure 12, paclitaxel group 10mg/kg, the tumor inhibition rate of human colon cancer HT-29 nude mice xenografts 69.11 %; Endo group 2.5mg/kg, the tumor inhibition rate of human colon cancer HT-29 nude mice xenografts was 33.48%; HM-3 group 1.5mg/kg, transplanted to human colon cancer HT-29 nude mice The tumor inhibition rate was 52.59%; the tumor inhibition rate of human colon cancer HT-29 nude mice was 55.98%, 45.45%, 37.05%, and therefore, mPEG-SC 2 was high, medium and low dose groups.
• the growth inhibition test of k- HM-3 on human colon cancer HT-29 nude mice showed that compared with the negative control group, the 36.7 mg/kg peptide group had significant growth in human colon cancer HT-29 xenografts. Inhibition.
• Example 17 mPEG-SC 2 . k -HM-3 inhibition of human ovarian cancer SK-OV-3 tumor growth in nude mouse xenograft human trials in the logarithmic phase of ovarian cancer SK-OV-3 cell line, prepared under sterile conditions 5xl0 7 / ml cell suspension, The mice were inoculated subcutaneously in the right axilla with 0.1 ml. The nude mice xenografts were measured for the diameter of the transplanted tumor using a vernier caliper, and the animals were randomly divided into groups after the tumors were grown to 100-200 mm 3 . The effect of the anti-tumor effect of the test substance was dynamically observed using a method of measuring the tumor diameter.
• the number of measurements of tumor diameter was measured once every 2 days.
• the administration methods were all injected by tail vein.
• the negative control group was injected with the same amount of normal saline once a day; the cisplatin group was 10 mg/kg once a week; the Endo group was 2.5 mg/kg, once a day; the polypeptide high, medium and low groups were 6, 3 respectively. , 1.5mg/kg, once a day.
• the mice were sacrificed and the tumor pieces were surgically removed and weighed.
• mPEG-SC 2 Inhibitory effect of k- HM-3 on xenograft tumor growth in human ovarian cancer SK-OV-3 nude mice
• HM-3 low results See Table 12 and Figure 13, cisplatin group 10mg/kg, inhibition rate of human ovarian cancer SK-OV-3 nude mice xenografts 76. 13%; Endo group 2.5mg/kg, the tumor inhibition rate of human ovarian cancer SK-OV-3 nude mice xenografts was 31.98%; HM-3 group 1.5mg/kg, for human ovarian cancer SK The tumor inhibition rate of -OV-3 nude mice xenografts was 49.49%. The tumor inhibition rate of human ovarian cancer SK-OV-3 nude mice was 50.40%, 44.62%, in the high, middle and low dose groups.
• 1 ml was inoculated subcutaneously to the right armpit of nude mice.
• Nude mouse transplanted tumors with vernier calipers to measure the diameter of the transplanted tumor, to be swollen Animals were randomized after tumor growth to 100-200 mm3 .
• the method of measuring the tumor diameter is used to dynamically observe the antitumor effect of the test substance.
• the number of measurements of tumor diameter was measured once every 2 days.
• the administration methods were all injected by tail vein.
• the negative control group was injected with the same amount of normal saline once a day; the paclitaxel group was 10 mg/kg once a week; the Endo group was 2.5 mg/kg, once a day; the high, medium and low peptides were 6, 3, respectively.
• mice were sacrificed and the tumor pieces were surgically removed and weighed. Inhibitory effect of mPEG-SC 20k- HM-3 on human endometrial cancer HHUA nude mouse xenograft tumor growth
• paclitaxel group 10mg/kg the tumor inhibition rate of human endometrial cancer HHUA nude mice xenografts was 75.55%; The degree of tumor inhibition of the endometrial cancer HHUA nude mice xenografts was 34.98%; the HM-3 group 1.5 mg/kg, the human endometrial cancer HHUA nude mice xenografts The tumor inhibition rate was 54.49%.
• the tumor inhibition rate of human endometrial cancer HHUA nude mice was 62.47%, 53.65%, 51.38% in high, medium and low dose groups. Therefore, mPEG-SC 2 .
• Example 19 Inhibition of xPEG-SC 20k- HM-3 on human cervical cancer HeLa nude mouse xenograft tumor growth
• the human cervical cancer HeLa cell line in logarithmic growth phase was prepared under aseptic conditions. 10 7 /ml cell suspension, The mice were inoculated subcutaneously in the right axilla with 0.1 ml.
• the nude mice xenografts were measured for the diameter of the transplanted tumor using a vernier caliper, and the animals were randomly divided into groups after the tumors were grown to 100-200 mm 3 .
• the effect of the anti-tumor effect of the test substance was dynamically observed using a method of measuring the tumor diameter. The number of measurements of tumor diameter was measured once every 2 days.
• the administration methods were all injected by tail vein.
• the negative control group was injected with the same amount of normal saline once a day; the paclitaxel group was 10 mg/kg once a week; the Endo group was 2.5 mg/kg, once a day; the high, medium and low peptides were 6, 3, respectively. 1.5 mg/kg, once a day.
• the mice were sacrificed and the tumor pieces were surgically removed and weighed.
• Negative control 1 once daily 23. 11 8 starting material 23.35 8 1.236 a paclitaxel 10 once every two days 24.36 6 23.47 6 0.428 65.37% grace 2.5 once daily 23.54 6 23.28 6 0.796 35.57%
• HM-3 low results See Table 14 and Figure 15, 16, paclitaxel group 10mg/kg, the tumor inhibition rate of human cervical cancer HeLa nude mice xenografts is 65.37 %; Endo group 2.5mg/kg, the tumor inhibition rate of human cervical cancer HeLa nude mice xenografts was 35.57%; HM-3 group 1.5mg/kg, anti-tumor of human cervical cancer HeLa nude mice xenografts The rate of 57.25% was high in the high, medium and low dose groups. The tumor inhibition rate of human cervical cancer HeLa nude mice was 82.07%, 74.11%, 63.32%. Therefore, mPEG-SC 2 . The growth inhibition test of k- HM-3 on human cervical carcinoma HeLa nude mice showed that compared with the negative control group, the 36.7 mg/kg peptide group significantly inhibited the growth of human cervical carcinoma HeLa xenografts.
• Example 20 Inhibition of mPEG-SC 20k- HM-3 on human prostate cancer DU-145 nude mouse xenograft tumor growth
• the human prostate cancer DU-145 cell line in logarithmic growth phase was prepared under aseptic conditions. 5 x l 0 7 /ml cell suspension was inoculated into the right axilla of nude mice with 0.1 ml. Nude mouse transplanted tumors with vernier calipers to measure the diameter of the transplanted tumor, to be swollen Animals were randomized after tumor growth to 100-200 mm3 . The method of measuring the tumor diameter is used to dynamically observe the antitumor effect of the test substance. The number of measurements of tumor diameter was measured once every 2 days.
• the administration methods were all injected by tail vein.
• the negative control group was injected with the same amount of normal saline once a day; the cisplatin group was 10 mg/kg once a week; the Endo group was 2.5 mg/kg, once a day; the polypeptide high, medium and low groups were 6, 3 respectively. , 1.5mg/kg, once a day.
• the mice were sacrificed and the tumor pieces were surgically removed and weighed.
• cisplatin group 10mg/kg, the tumor inhibition rate of human prostate cancer DU-145 nude mice xenografts is 70.60%
• the enzymatic group 2.5mg/kg, the tumor inhibition rate of human prostate cancer DU-145 nude mice xenografts was 3 1.25%; HM-3 group 1.5mg/kg, transplantation of human prostate cancer DU-145 nude mice
• the tumor inhibition rate was 56.36%.
• the tumor inhibition rate of human prostate cancer DU-145 nude mice was 77.46%, 67.48%, 57.87% in the high, medium and low dose groups.
• Example 21 Inhibition of mPEG-SC 20k- HM-3 on human bladder cancer HT 1376 nude mouse xenograft tumor growth
• the human bladder cancer HT 1376 cell line in logarithmic growth phase was prepared and prepared under aseptic conditions.
• Xl 0 7 /ml cell suspension was inoculated subcutaneously in the right axilla of nude mice with 0.1 ml.
• the nude mice xenografts were measured for the diameter of the transplanted tumor using a vernier caliper, and the animals were randomly divided into groups after the tumors were grown to 100-200 mm 3 .
• the effect of the anti-tumor effect of the test substance was dynamically observed using a method of measuring the tumor diameter.
• the number of measurements of tumor diameter was measured once every 2 days.
• the mode of administration was uniformly injected into the tail vein.
• the negative control group was injected with the same amount of normal saline once a day; the paclitaxel group was 10 mg/kg once a week; the Endo group was 2.5 mg/kg, once a day; the high, medium and low peptides were 6, 3, respectively. 1.5 mg/kg, once a day.
• the mice were sacrificed and the tumor pieces were surgically removed and weighed.
• Negative control one daily - - times 21.71 10 21.32 10 1.018 one paclitaxel 10 two days one - time 21.39 6 21.65 5 0.327 67.88% grace 2.5 daily - - time 21.52 21.36 6 0.698 3 1.43%
• Example 22 Inhibition of mPEG-SC 20k- HM-3 on human testicular cancer 5637 nude mouse xenograft tumor growth
• the human testicular cancer 5637 cell line in logarithmic growth phase was prepared under aseptic conditions to prepare 5 x l 0 7 / ml cell suspension, inoculated in the right armpit of nude mice with 0.1 ml.
• the nude mice xenografts were measured for the diameter of the transplanted tumor using a vernier caliper, and the animals were randomly divided into groups after the tumors were grown to 100-200 mm 3 .
• the method of measuring the tumor diameter is used to dynamically observe the antitumor effect of the test substance. The number of measurements of tumor diameter was measured once every 2 days.
• mice were sacrificed and the tumor pieces were surgically removed and weighed. Table 17. Inhibition of mPEG-SC 20k- HM-3 on human testicular cancer 5637 xenograft tumor growth with starting body terminal body weight
• Dosing frequency inhibition rate ( ⁇ 1 ⁇ 4) ( mg / weight (g ) ( g )
• HM-3 low results See Table 17 and Figure 20, cisplatin group 10mg/kg, tumor inhibition rate of human testicular cancer 5637 nude mice xenografts 70.74% The degree of tumor inhibition of the implanted tumor of 5637 nude mice was 30.02%, and that of the HM-3 group was 1.5mg/kg, which inhibited the tumor growth rate of human testicular carcinoma 5637 nude mice.
• the tumor inhibition rate of the high-, medium-, and low-dose peptides in human testicular cancer 5637 nude mice was 60.20%, 51.32%, and 50.99%. Therefore, mPEG-SC 2 .
• Example 25 Inhibition of xenograft tumor growth in sarcoma HT-1080 nude mice by mPEG-SC 20k- HM-3
• the sarcoma HT-1080 cell line in logarithmic growth phase was prepared under aseptic conditions to prepare 5 x l 0 7 / ml cell suspension, inoculated in the right armpit of nude mice with 0.1 ml.
• the nude mice xenografts were measured for the diameter of the transplanted tumor using a vernier caliper, and the animals were randomly divided into groups after the tumors were grown to 100-200 mm 3 .
• the method of measuring the tumor diameter is used to dynamically observe the antitumor effect of the test substance.
• the number of measurements of tumor diameter was measured once every 2 days.
• the administration methods were all injected by tail vein. Negative control group injection equivalent Normal saline once a day; 15 mg/kg in the cyclophosphamide group, once a week; the polypeptide was administered once a day at 3 mg/kg. After the end of the administration, the mice were sacrificed and the tumor pieces were surgically removed and weighed.
• results See Table 18 and Figure 21, 10 mg/kg of cyclophosphamide group, 74.15% for sarcoma HT-1080 nude mice xenografts; 1.5 mg/kg for HM-3 group, for human sarcoma HT-1080
• the tumor inhibition rate of nude mice xenografts was 59.24%; the tumor inhibition rate of dermatophys HT-1080 nude mice xenografts was 65.04%. Therefore, mPEG-SC 2 .

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