WO2013075600A1 - 一种聚乙二醇修饰的整合素阻断剂hm-3及其应用 - Google Patents

一种聚乙二醇修饰的整合素阻断剂hm-3及其应用 Download PDF

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WO2013075600A1
WO2013075600A1 PCT/CN2012/084788 CN2012084788W WO2013075600A1 WO 2013075600 A1 WO2013075600 A1 WO 2013075600A1 CN 2012084788 W CN2012084788 W CN 2012084788W WO 2013075600 A1 WO2013075600 A1 WO 2013075600A1
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tumor
once
human
mpeg
group
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PCT/CN2012/084788
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English (en)
French (fr)
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徐寒梅
常海民
康志安
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Xu Hanmei
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Priority to AU2012343020A priority Critical patent/AU2012343020B2/en
Priority to KR1020147016557A priority patent/KR20140096373A/ko
Priority to US14/359,462 priority patent/US20140329759A1/en
Priority to KR1020197020874A priority patent/KR102106485B1/ko
Priority to EP12851929.5A priority patent/EP2784093B1/en
Priority to IN4482CHN2014 priority patent/IN2014CN04482A/en
Publication of WO2013075600A1 publication Critical patent/WO2013075600A1/zh
Priority to US15/332,539 priority patent/US20170100489A1/en

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    • 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, 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 .

Abstract

本发明公开了一种经聚乙二醇修饰的多肽及其在制备治疗肿瘤药物中的应用，所述多肽具有抑制肿瘤血管生成的功能，是一种具有整合素亲和性和结合能力的整合素阻断剂。所述经修饰的多肽可用于实体肿瘤的治疗。

Description

一种聚乙二醇修饰的整合素阻断剂 HM-3及其应用
技术领域
本发明涉及药物领域， 具体涉及具有抑制肿瘤血管生成、 具有整合素亲和性和结合能 力的一种整合素阻断剂， 此阻断剂是一种聚乙二醇修饰多肽， 该整合素阻断剂聚乙二醇修 饰多肽可用于实体肿瘤的治疗。 背景技术
研究表明， 实体瘤的生长依赖于新说生血管生成， 新生血管不仅可以提供肿瘤所需要的 营养和氧气， 排泄代谢产物， 而且是远处转移的途径。 因此， 阻断新生血管形成可能成为 书
阻止肿瘤生长和转移的手段， 从而激发了对促血管生成分子和抗血管生成分子的广泛研究。 相比传统肿瘤治药物疗， 肿瘤血管生成抑制剂最大优点在于： ①选择性作用于血管内 皮细胞， 全身毒副作用小； ②靶细胞为血管内皮细胞， 药物易从血液中到达与之接触发生 作用； ③血管内皮细胞无或少突变， 不易产生耐药性， 可长期用药； ④可与放化疗方法联 合应用并减轻后者的毒副反应。
目前， 国际上开发出的整合素阻断剂已进入 II期临床。 而我国尚未有相似或同类产品 进入市场， 非常有必要开发我国自主知识产权的此类药物。 ZL2005100403785 高效抑制血 管生成多肽及其制备方法和应用， 介绍几种整合素抑制剂， 其一为整合素阻断剂多肽序列 为： Ile-Val- Arg- Arg-Ala-Asp- Arg-Ala- Ala-Val-Pro-Gly-Gly-Gly-Gly- Arg-Gly-Asp， 该序列包 含了整合素配体序列 （ Gly-Gly-Gly-Gly-Arg-Gly-Asp ) 和新生血管抑制序列 ( Ile-Val-Arg-Arg-Ala-Asp-Arg-Ala-Ala-Val-Pro )， 其中整合素配体序列中含有 RGD 序列 ( Arg-Gly-Asp ) , 整合素阻断剂多肽序列可以有效地结合于肿瘤特异性表达的整合素亚型， 并且该序列中含有新生血管抑制序列， 从而抑制肿瘤新生血管形成， 进而达到抑制肿瘤生 长和转移的效果。前期研究表明其作用靶点为整合素 α ^3和 α5 7，但主要的结合靶点仍为 整合素 οαφ3。 该多肽经反复体内外活性评价证实具有较好的抑瘤效果， 能够显著抑制内皮 细胞迁移、 抑制肿瘤新生血管生成， 从而抑制肿瘤的生长。 然而上述多肽的半衰期短， 拟 用临床人的给药是每天静脉滴注， 这给患者带来了一定的痛苦。
文献报道中， 对分子结构进行修饰或改造是解决半衰期较短、 需连续给药问题的 常用方法， 其中以化学修饰应用最为广泛， 常用的化学修饰剂有聚乙二醇 (polye thylene glycol, PEG). 葡聚糖、 聚氨基酸、 聚酸酐等。 PEG具有无毒、 无免疫原性、 水溶性好的特 点， 被美国食品与药物管理局 (FDA)认可作为药品的辅助原料和修饰剂。 蛋白质类药物经 PEG修饰后， 分子量增加， 肾小球的滤过率减少， PEG的屏障作用保护了蛋白质不易被蛋 白水解酶水解， 同时减少了中和抗体的产生， 这些均有助于蛋白质类药物生物半衰期的延 长。 目前已有多种 PEG修饰的蛋白质类药物上市销售。但 PEG修饰同时也可能影响蛋白质 的生物学活性， 其影响大小与修饰剂、 修饰条件及蛋白质本身的性质有关。 对于具体的蛋 白质， 其最佳修饰需通过制备 PEG修饰的蛋白质及生物活性研究来决定。 合成小分子多肽 的 PEG修饰研究起步较晚， 但已引起不少研究者的关注。 发明内容 发明目的
本 发 明 针 对 mPEG-SC-Ile-Val-Arg-Arg-Ala-Asp-Arg-Ala-Ala-Val-Pro-Gly-Gly-Gly- Gly-Arg-Gly-Asp做了进一步研究，发现其在降低给药频率的情况下对多种肿瘤有治疗作用。 技术方案
一种聚乙二醇修饰的整合素阻断剂 HM-3 , 其中整合素阻断剂的序列为 mPEG-SC-Ile-Val-Arg-Arg-Ala-Asp-Arg-Ala-Ala-Val-Pro-Gly-Gly- Gly-Gly -Arg-Gly-Asp, 其 特征在于所述的 mPEG-SC的分子量范围为 500-20000。
作为一种优化方式所述的一种聚乙二醇修饰的整合素阻断剂 HM-3 , 其特征在于所述 的 mPEG-SC (单甲氧基聚乙二醇-琥珀酰亚胺碳酸酯)的分子量为 20000。
一种聚乙二醇修饰的整合素阻断剂 HM-3 在制备治疗肿瘤药物中的应用， 其特征在于 所述的肿瘤为起源于人的胃部、 皮肤、 头颈部、 甲状腺、 胰腺、 肺脏、 食管、 乳腺、 肾脏、 胆囊、 结肠 /直肠、 卵巢、 子宫、 子宫颈、 前列腺、 膀胱、 睾丸的原发 /继发的癌或肉瘤。 有益效果
1、 为了延长多肽 lie- Val-Arg-Arg-Ala- Asp-Arg-Ala-Ala-Val-Pro-Gly- Gly- Gly- Gly- Arg-Gly-Asp的半衰期， 我们采用不同分子量的聚乙二醇（PEG)对此多肽进行了修饰， 发现 mPEG-SC2Qk-HM-3具有延长了 HM-3半衰期， 但又不影响其体内外活性的特点。 一个 多肽的修饰产物属于一个新型分子， 往往与修饰前的分子在活性上产生不同的效果。 本发 明针对上述整合素阻断剂 mPEG-SC2Qk-HM-3对多种肿瘤有治疗作用进行了大量的体内外活 性研究， 发现给药频率降低的情况下， mPEG-SC2Qk-HM-3对抑制多种肿瘤生长保持了很好 的活性， 拓展了其社会价值和经济价值。 2、 研究发现， 序列 Ile-Val-Arg-Arg-Ala-Asp-Arg-Ala-Ala-Val-Pro具有抑制肿瘤血管生 成的作用。 精氨酸 -甘氨酸-天冬氨酸 （RGD) 序列是整合素的一个重要配体， 因此， 含有 RGD序列的多肽 Gly-Gly-Gly-Gly-Arg-Gly-Asp也能够特异性的识别整合素。本发明的整合 素 阻 断 剂 多 肽 是 在 具 有 抑 制 血 管 生 成 作 用 的 序 列 Ile-Val-Arg-Arg-Ala-Asp-Arg-Ala-Ala-Val-Pro的 C端连接上与整合素家族具有亲和性和结合 能力的含有精氨酸 -甘氨酸-天冬氨酸 （RGD) 的 Gly-Gly-Gly-Gly-Arg-Gly-Asp序列， 构建 了一种与整合素有亲和性和结合能力的多肽。 同时， 在该整合素阻断剂多肽的 N端特别优 化 进 行 了 聚 乙 二 醇 修 饰 ， 最 终 优 化 的 序 列 为 ： mPEG-SC20k-Ile-Val-Arg-Arg-Ala-Asp-Arg-Ala-Ala-Val-Pro-Gly-Gly-Gly-Gly-Arg-Gly-Asp, 其 含有 PEG和 18个氨基酸的多肽， 分子中 RGD序列具有整合素亲和性和结合能力， 研究表 明其起作用靶点为整合素 aVW和 Cd^结合， 但主要的结合靶点仍为整合素 aVW , 并且该 序列中含有新生血管抑制序列， 从而抑制肿瘤新生血管形成， 进而达到抑制肿瘤生长和转 移的效果。 聚乙二醇 （PEG) 是一类具有独特理化性质的大分子聚合物， 它具有良好的生 物相容性， 无毒、 无抗原性。 蛋白质或多肽药物经 PEG修饰后， 其主要的生物学功能保持 不变， 并且经 PEG修饰能赋予蛋白质和多肽类药物多种优良性能： （1)增加稳定性， 延长血 浆半衰期； （2)降低免疫原性和抗原性； （3)降低毒副作用； （4)减小被水解酶降解的可能性、 降低被肾脏清除的速率； （5)改善药物体内分布和动力学行为等。
聚乙二醇 （mPEG-SC2Qk) 修饰后多肽的靶点不变， 延长多肽分子的体内半衰期、 清除 率低、 降低了免疫原性和抗原性， 并且抗肿瘤活性保持不变， 但是降低了给药频率， 修饰 后由原来每天给药一次， 变为每 2-3天给药一次。
发明人经过大量实验获知该整合素阻断剂体内实验具有明显的抗肿瘤效果， 并且副作 用少， 用量少成本低。 说明本发明设计的聚乙二醇修饰的整合素阻断剂多肽科学、 合理、 可行有效， 能作为制备治疗人类实体肿瘤的治疗药物， 为将来药物开发提供了新的思路和 前景， 具有显著的社会价值和市场价值。
修饰前多肽 HM-3的半衰期为 0.46h， 经 mPEG-SC2Qk修饰后的半衰期为 20.13h。 如下 表：
表 1 mPEG-SC2。k-HM-3和 HM-3的半衰期比较 Λ1/2β为半衰期）
药物 t！。 β (h) CL (L/h/kg) AUC0.m (mg/Uh) MRT0.∞ (h)
mPEG-SC20k-HM-3 20.13 ± 0.64 0.0071 ± 0.0012 4391.22 ± 3562.89 15.35 ± 1.07
HM-3 0.46 ± 0.12 1.38 ± 0.15 25.63 ± 9.76 0.036 ± 0.002 附图说明
附图 1流式细胞实验检测整合素阻断剂多肽与靶点的结合， 其中 a为第一次实验-， b 为重复实验；
附图 2整合素阻断剂多肽与 HM-3免疫原性比较；
附图 3整合素阻断剂多肽对人食管癌 Ecl09裸鼠异种移植肿瘤生长抑制作用； 附图 4整合素阻断剂多肽对人鼻咽癌 C E裸鼠异种移植肿瘤生长抑制作用； 附图 5整合素阻断剂多肽对人甲状腺癌 SW-579裸鼠异种移植肿瘤生长抑制作用； 附图 6整合素阻断剂多肽对人胃癌 MGC803裸鼠异种移植肿瘤生长抑制作用； 附图 7整合素阻断剂多肽对人胰腺癌 SW-1990裸鼠异种移植肿瘤生长抑制作用； 附图 8整合素阻断剂多肽对人肺癌 H460裸鼠异种移植肿瘤生长抑制作用；
附图 9整合素阻断剂多肽对人乳腺癌 MDA-MB-231裸鼠异种移植肿瘤生长抑制作用； 附图 10整合素阻断剂多肽对人胆囊癌 GBC-SD裸鼠异种移植肿瘤生长抑制作用； 附图 11整合素阻断剂多肽对人肾癌 A498裸鼠异种移植肿瘤生长抑制作用； 附图 12整合素阻断剂多肽对人结肠癌 HT-29裸鼠异种移植肿瘤生长抑制作用； 附图 13整合素阻断剂多肽对人卵巢癌 SK-OV-3裸鼠异种移植肿瘤生长抑制作用； 附图 14整合素阻断剂多肽对人子宫内膜癌 HHUA裸鼠异种移植肿瘤生长抑制作用； 附图 15整合素阻断剂多肽对人宫颈癌 HeLa裸鼠异种移植肿瘤生长抑制作用； 附图 16整合素阻断剂多肽对人宫颈癌 HeLa裸鼠异种移植肿瘤生长抑制作用剖取的肿 瘤实物图；
附图 17整合素阻断剂多肽对人前列腺癌 DU-145裸鼠异种移植肿瘤生长抑制作用； 附图 18整合素阻断剂多肽对人膀胱癌 HT1376裸鼠异种移植肿瘤生长抑制作用； 附图 19整合素阻断剂多肽对人膀胱癌 HT1376裸鼠异种移植肿瘤生长抑制作用剖取的 肿瘤实物图；
附图 20整合素阻断剂多肽对人睾丸癌 5637裸鼠异种移植肿瘤生长抑制作用； 附图 21整合素阻断剂多肽对肉瘤 HT-1080裸鼠异种移植肿瘤生长抑制作用； 具体实施方式
实施例 1 整合素阻断剂 HM-3
Ile-Val-Arg-Arg-Ala-Asp-Arg-Ala-Ala-Val-Pro-Gly-Gly-Gly-Gly-Arg-Gly-Asp的固相合成 方法， 其以 Fmoc- Ile-wang resin或 Fmoc- lie -CTC resin为起始原料， 然后用保护氨基酸依 次接二肽至十八肽， 接肽工作完成后充分洗涤， 然后切肽、 后处理即得 HM-3 粗品。 将粗 品进行纯化， 首先溶解， 然后用制备型高效液相经过两次纯化， 最后浓缩冻干得到纯品。 具体步骤如下：
一、 合成：
称取 Fmoc- lie -Wang resinl4.7g, 倒入 1L的玻璃砂芯反应柱中， 加入 CH2C12 147ml 使树脂充分膨胀。
脱帽： 加入六氢吡啶 / DMF的脱帽液 25ml, 密封后放入振荡器中反应 5分钟， 温度控 制在室温， 5分钟后将脱帽液抽干，中间用 DMF洗涤一次，然后再加入一次 20%的脱帽液 25ml 反应 15分钟；
洗涤： 将脱帽液抽干， 用 DMF洗涤树脂 6次， 抽干， 取 20颗树脂在小试管内， 加入验 色剂， 在 115°C加热 3分钟。
缩合： 称取保护氨基酸和 HOBt 2.025g， 溶于 15ml DMF和 2.33ml DIC, 然后倒入反应 釜中反应 1.5左右小时， 温度控制在 34°C左右。
洗涤： 将反应液抽干， 用 DMF洗涤树脂 3次， 抽干， 取 10-20颗树脂在小试管内， 加 入验色剂， 在 115°C加热 3-5分钟。
切肽： 把抽干的树脂装入 500mL的圆底烧瓶中， 加入 300mL90%的切割液 （按体积比 比计 三氟乙酸： 苯酚： 苯甲硫醚： EDT=90: 3： 3： 2： 2)， 密封反应 2小时， 用砂芯漏斗 将树脂与多肽分离。
后处理： 先加无水乙醚到切割液中将多肽析出， 然后离心,把清液倒掉,然后再用无水乙 醚洗涤多肽 6次， 抽干得多肽粗品 9.5g。
二、 纯化：
溶解： 精密称取 ID-18粗品， 加入适当的纯化水配置成 lOg/1的溶液， 超声搅拌至无颗 粒状澄清溶液。
过滤： 将 ID-18的溶液用沙芯过滤器 0.45um的混合滤膜过滤.
制备：
一次纯化
平衡： 制备柱用 5%乙腈 +95%三氟乙酸水溶液， 流速 80ml/min冲洗 10min。 上样： 用输液泵上样， 流速 80ml/min,并采集基线， 收取在紫外波长 220nm 吸收大于 200mv的溶液， 检测是否有样品冲出。
洗脱：
洗脱梯度：
Figure imgf000008_0001
收取在紫外波长 220nm吸收大于 200mv的溶液， 检测纯度大于 95%的合并作为峰顶， 待做二次分离纯化。
二次纯化
平衡： 制备柱用 5%乙腈 +95%醋酸水溶液， 流速 80ml/min冲洗 lOmin
上样： 将一次收到的峰顶旋转蒸发掉有机溶剂后用输液泵上样， 流速 80ml/min,并采集 基线， 收取在紫外波长 220nm吸收大于 200mv的溶液， 检测是否有样品冲出。
洗脱：
洗脱梯度：
Figure imgf000008_0002
收取在紫外波长 220nm吸收大于 200mv的溶液， 通过检测纯度大于 99%的作为合格。 浓缩、 过滤、 冻干： 将合格溶液用旋转蒸发仪 37°C减压浓缩， 除去残留溶剂和注射用水。 最后用 0. 22um滤膜过滤， 滤液装入冻干盘中， 用冷冻干燥机进行冷冻干燥， 得纯品。
实施例 2 聚乙二醇修饰多肽的步骤
mPEG-SC2()K与 HM-3的反应
分别称取 2gmPEG-SC2()K和 106.24mgHM-3 (摩尔比为 1.5: 1 )置于 40ml-100ml配好 的 PH 5-8.5的 PBS缓冲溶液中， 在 4 °C条件下过夜， 使其进行反应。 PEG-SC500-20000都 可按实施例 2进行连接反应合成修饰多肽。 实施例 3 分离纯化步骤
一、 分离
对反应后的样品应用半制备型高效液相 （HPLC， BIO-RAD) 进行纯化， 纯化条件为： 流动相： ACN (+0.1% TFA)、 H20 (+0.1% TFA); ACN线性梯度： 40%-95%； 流速： 2 ml/min; 运行时间： 12 min;
上样量： 1.0 ml; 检测波长： 220 nm。
半制备色谱柱： YMC， 250 mm lO mm ( 5 μιη ±真料）。
在目的峰出峰过程中， 用离心管收集产物。
二、 纯化
将经过 HPLC收集的产物首先于 -70°C低温冰箱预冻过夜， 之后于提前遇冷的冷冻干燥 机冻干， 直到目测全为白色粉末为止 （30 h左右） 。 收获冻干产物， 称量并记录产物重量 后于 -20°C冰箱保存， 并进行鉴定。
1.产物的纯度分析
将冻干后的产物， 通过分析型高效液相色谱分析纯度， 分析条件为：
流动相： ACN (+0.1% TFA) 、 H2O(+0.1% TFA) ； ACN线性梯度： 10%-100%；
、流速： 1 ml/min; 运行时间： 15 min;
上样量： 20 μΐ; 检测波长： 220 nm。
分析型色谱柱： 北京创新通恒， 250 mmx4.6 mm ( 5 μιη填料）。
2. 修饰产物 SDS-PAGE分析
基本操作参考《分子克隆（第二版）》。 浓缩胶浓度为 5 %， 分离胶浓度为 10%， 浓缩电 压 80伏， 分离电压 120伏。 电泳结束后样品条带先进行 Bal2.染色， 对含有 PEG的部分进 行染色； marker用考马斯亮兰 R250对蛋白部分染色。染色完毕放入脱色液中至本底透明后 扫描分析。
下面的实施例以 mPEG20000修饰的整合素阻断剂多肽（mPEG-SC2。k-HM-3 )为例， 进行说 明。 实施例中所说的整合素阻断剂多肽即是 mPEG20000修饰的整合素阻断剂多肽
实施例 4
mPEG-SC20k-HM-3在大鼠体内药代动力学研究
SD大鼠， 随机分为 6组， 雌雄各半。 取 3组尾静脉分别给予整合素阻断剂多肽高剂量 52 mg/kg (折合 HM-3 4.2 mg/kg),中剂量 26 mg/kg (折合 HM-3 2.1mg/kg),低剂量 13 mg/kg (折合 HM-3 1.05 mg/kg )， 另外 3组分别注射 HM-3 高剂量 4.2 mg/kg， 中剂量 2.1mg/kg， 低剂量 1.05 mg/kg， 各组大鼠分别于给药后 0.5h、 lh、 2h、 3h、 6h、 12h、 24h、 48h、 72h、 96h、 108h、 132h由眼眶静脉丛采血， 每点采全血 0.5 ml， 肝素抗凝， 12000 rpm/2min离心 分离血浆， 吸取上清血浆各 200 μΐ与 80°C预热的 PBS ( 0.05M pH7.4 ) 缓冲液 600 μΐ 混匀， 80°C水浴 30 min， 12000 rpm离心 2 min， 取上清液， -20°C低温保存备用。 待室温溶解后， 用 ELISA方法测定整合素阻断剂多肽血药浓度。
表 1 mPEG-SC2。k-HM-3和 HM-3在 SD大鼠体内的药代动力学参数比较 ι/2β为半衰期， 为血浆清除率， ^f/C为曲线下面积， 为平均滞留时间）
Figure imgf000010_0001
由表 1可知， mPEG-SC2。k-HM-3与 HM-3相比， 半衰期有显著延长， 血浆清除率明显降低。 以上实验数据均证明 PEG修饰能够显著改善蛋白多肽药物在大鼠体内的药代动力学特征。
实施例 5
流式细胞试验分析 mPEG-SC2。k-HM-3与靶点的结合
( 1 ) 肿瘤细胞 Bel-7402和 MCF-7在 24孔板中培养至 80%的融合后， 胰酶消化收集， 用冰预冷的 PBS洗 2次， 在标记前将细胞用含 1% BSA的 PBS重悬 30 min。
( 2 )用 2 μΐ鼠抗人 ανβ3 功能阻断单克隆抗体 (1.0 g l, 1 :200) 和 2 μΐ鼠抗人 α5β 1功 能阻断单克隆抗体 θ μ8/μ1, 1 :200)与细胞悬液在 4°C孵育 1.5h。
( 3 ) 标记后将细胞收集， 并用冰预冷的 PBS洗涤 2次， 之后用 lOO l FITC标记的修 饰多肽 mPEG-SC2()k-HM-3 (2 mg/ml)与细胞悬液在 4°C孵育 1.5h。
( 4 ) 标记后， 将细胞收集并用冰预冷的 PBS洗涤 2次， 之后用 400 μΐ PBS重悬， 用 流式细胞仪分析， FITC荧光用 FL1通道检测荧光强度。
由图 1显示， mPEG-SC2。k-ffl-3能够和整合素《 /^和《5/3 结合， 但主要的结合靶点仍为整 合素 ανβ3。 表明， 经 PEG修饰后 HM-3的主要作用靶点未发生改变， 活性位点未被 PEG所 覆盖。
实施例 6
mPEG-SC20k-HM-3和 HM-3免疫原性检测
BALB/c白鼠，随机分为 2组，雌雄各半，尾静脉分别给予 36 mg/kg的 mPEG-SC2。k-HM-3 和 3.0 mg/kg的 HM-3。 连续给药 8周， 于 1-12周眼眶静脉丛定点取血每周一次， 12000rpm 离心 2 min, 分离血浆取上清液， -20°C低温保存备用。 待室温溶解后， 取 0.1 ml上清液间 组别设置：
第一组有效给药剂量的 HM-3 3.0 mg/kg， 给药： 1天 1次， 6只动物雌雄各半； 第二组有效给药剂量的 mPEG-sc2。k -匪 -336 mg/kg, 给药： 2天 1次， 6只动物雌雄各半。 由图 2显示， HM-3组给药第 3周便有抗体产生， 而 mPEG-SC2。k-HM-3组给药第 5周才检 测到较低滴度的抗体。 且各时间点整合素阻断剂多肽组抗体滴度明显低于 HM-3 组， 停药 后抗体滴度逐渐下降， 到第 12周整合素阻断剂多肽组抗体已检测不到。 说明 PEG修饰可 显著降低 HM-3体内免疫原性。
实施例 7
mPEG-SC20k-HM-3对人食管癌 EC109裸小鼠异种移植肿瘤生长抑制试验 取生长旺盛期的瘤组织剪切成 1.5mm3左右， 在无菌条件下， 接种于裸小鼠右侧皮下。 小鼠移植瘤用游标卡尺测量移植瘤直径， 待肿瘤生长至 100-200mm3后动物随机分组。 使用 测量瘤径的方法， 动态观察被试多肽抗肿瘤的效应。 肿瘤直径的测量次数为每 2天 1次， 每次测量同时还需称量鼠重。 实验组尾静脉注射多肽， 每三天 1 次， 阴性组同时给等量生 理盐水。 肿瘤体积计算公式： 末动物数终
TV=0.52 X a X b2 其中 a、 b分别表示长宽。 根据测量的结果计算出相对肿瘤体积。 抗肿瘤活性的评价指 标为相对肿瘤增殖率 T/C(%；)， 计算公式如下：
T/C(%)=TRTV/CRTV X 100%
TRTV: 治疗组 RTV; CRTV: 阴性对照组 RTV
表 2. mPEG-SC20k-HM-3对人食管癌 EC109裸鼠异种移植肿瘤生长的抑制作用 重
瘤重（g) 抑瘤率
12 0.926
Figure imgf000011_0001
88 00..227766 70.19% 每天一次 2211..5555 88 2211..2266 88 00..660022 35.0% HM-3 1.5 三天一次 21.42 8 20.74 8 0.365 60.57% mPEG-SC
-HM-3 36.7 三天一次 22.20 8 21.75 8 0.183 62.24%* l¾
mPEG-SC
-HM-3 18.75 三天一次 20.45 8 19.93 8 0.318 55.66% 中
mPEG-SC
-HM-3 9.38 三天一次 21.22 8 20.38 8 0.406 50.16% 低 结果： 见表 2和图 3， 紫杉醇组 10mg/kg， 对人食管癌 Ecl09裸小鼠移植瘤的抑瘤率为 70.19%； 恩度组 2.5mg/kg， 对人食管癌 Ecl09裸小鼠移植瘤的抑瘤率为 35.0%; HM-3组 1.5mg/kg， 对人食管癌 Ecl09裸小鼠移植瘤的抑瘤率为 60.57%; 多肽高、 中、 低剂量组对 人食管癌 Ecl09裸小鼠移植瘤的抑瘤率达 62.24%， 55.66%, 50.16%。 mPEG-SC20k-HM-3对人食管癌 EC109裸小鼠移植瘤生长抑制试验结果表明，与阴性对照组相 比， 多肽 36.7mg/kg组对人食管癌 Ecl09移植瘤的生长有显著性的抑制作用。 *Ρ〈0· 05 (和 阴性组比较有显著性差异）
实施例 8
mPEG-SC20k-HM-3对人鼻咽癌 C E裸小鼠异种移植肿瘤生长抑制试验 取生长旺盛期的瘤组织剪切成 1.5mm3左右， 在无菌条件下， 接种于裸小鼠右侧皮下。 小鼠移植瘤用游标卡尺测量移植瘤直径， 待肿瘤生长至 100-200mm3后动物随机分组。 使用 测量瘤径的方法， 动态观察被试多肽抗肿瘤的效应。 肿瘤直径的测量次数为每 2天 1次， 每次测量同时还需称量鼠重。 实验组尾静脉注射多肽， 每三天 1 次， 阴性组同时给等量生 理盐水。 肿瘤体积计算公式：
TV=0.52 X a X b2 其中 a、 b分别表示长宽。 根据测量的结果计算出相对肿瘤体积。 抗肿瘤活性的评价指 标为相对肿瘤增殖率 T/C(%；)， 计算公式如下：
T/C(%)=TRTV/CRTV X 100%
TRTV: 治疗组 RTV; CRTV: 阴性对照组 RTV 表 3. mPEG-SC20k-HM-3对人鼻咽癌 CNE裸鼠异种移植肿瘤生长的抑制作用 剂量 起始体 终末体重
给药频率 抑瘤率 ( mg/kg ) 重 （g ) ( g )
Figure imgf000013_0001
阴性对照 一 每天一次 22.78 12 22.37 12 1.201 一 顺铂 10 每周两次 22.35 8 22.33 7 0.320 73.36% 恩度 2.5 每天一次 22.43 8 22.39 8 0.757 37.01%
HM-3 1.5 三天一次 22.60 8 22.85 8 0.468 61.03% mPEG-SC20k-
36.7 三天一次 22. 16 8 22. 15 8 0.382 68.22%* HM-3高
mPEG-SC20k-
18.75 三天一次 22.75 8 24.00 8 0.406 66. 19% HM-3中
mPEG-SC20k-
9.38 三天一次 23.02 8 23.70 8 0.537 55.32% HM-3低
起始动物数
结果： 见表 3 和图 4， 顺铂组 10mg/kg， 对人鼻咽癌 C E裸小鼠移植瘤的抑瘤率为 73.36% , 但对实验动物的体重具有显著性的影响； 恩度组 2.5mg/kg， 对人鼻咽癌 CNE裸小 鼠移植瘤的抑瘤率为 37.01% ; HM-3组 1.5mg/kg， 对人鼻咽癌 C E裸小鼠移植瘤的抑瘤率 为 61.03% ;多肽高、中、低剂量组对人鼻咽癌 CNE裸小鼠移植瘤的抑瘤率为 68.22%， 66. 19%， 55.32% , 对实验动物体重无显著性影响。 末动物数终 mPEG-SC20k-HM-3对人鼻咽癌 C E裸小鼠移植瘤生长抑制试验结果表明， 与阴性对照组相 比， 多肽 36.7mg/kg组对人鼻咽癌 C E移植瘤的生长有显著性的抑制作用； 与阳性对照顺 铂组相比， 对实验动物的体重没有影响， 未见明显的毒副反应。 *P〈0.05 (和阴性组比较有 显著性差异） 实施例 9
mPEG-SC2。k-HM-3对人甲状腺癌 SW-579裸小鼠异种移植肿瘤生长抑制试验 取生长旺盛期的瘤组织剪切成 1.5mm3左右， 在无菌条件下， 接种于裸小鼠右侧皮下。 小鼠移植瘤用游标卡尺测量移植瘤直径， 待肿瘤生长至 100-200mm3后动物随机分组。 使用 测量瘤径的方法， 动态观察被试多肽抗肿瘤的效应。 肿瘤直径的测量次数为每 2天 1次， 每次测量同时还需称量鼠重。 实验组尾静脉注射多肽， 每三天 1 次， 阴性组同时给等量生 理盐水。 肿瘤体积计算公式：
TV=0.52 X a X b2 其中 a、 b分别表示长宽。 根据测量的结果计算出相对肿瘤体积。 抗肿瘤活性的评价指 标为相对肿瘤增殖率 T/C(%；)， 计算公式如下:
T/C(%)=TRTV/CRTV X 100%
TRTV: 治疗组 RTV; CRTV: 阴性对照组 RTV
表 4. mPEG-SC20k-HM-3对人甲状腺癌 SW-579裸鼠异种移植肿瘤生长的抑制作用 剂量 起始体 终末体重
给药频率 抑瘤率
Figure imgf000014_0001
( mg/kg) 重 （g) ( g) 阴性对
一 每天一次 21.34 12 21.23 12 1.253 一 照、
5-Fu 10 每天一次 21.56 8 21.32 7 0.320 74.50% 恩度 2.5 每天一次 21.67 8 20.39 8 0.850 32.20%
HM-3 1.5 每天一次 22.76 起始动物数 8 22.70 8 0.530 57.70% mPEG-SC20k
36.7 三天一次 22.32 8 21.76 8 0.406 67.63%* -HM-3高
mPEG-SC20k
18.75 三天一次 22.90 8 22.42 8 0.494 60.56% -HM-3中
mPEG-SC20k
9.38 三天一次 21.58 8 21.28 末动物数终 8 0.521 58.42% -HM-3低 结果： 见表 4和图 5， 5-Fu ( 5-氟尿嘧啶） 组 10mg/kg， 对人甲状腺癌 SW-579裸小鼠 移植瘤的抑瘤率为 74.50%， 但 5-Fu毒性较大， 动物体重下降， 实验过程中动物有死亡； 恩 度组 2.5mg/kg，对人甲状腺癌 SW-579裸小鼠移植瘤的抑瘤率为 32.20%; HM-3组 1.5mg/kg， 对人甲状腺癌 CNE裸小鼠移植瘤的抑瘤率为 57.70% ; 多肽高、 中、低剂量组对人甲状腺癌 SW-579裸小鼠移植瘤的抑瘤率达 67.63%， 60.56%, 58.42%, 对裸鼠体重没有显著性影响。 mPEG-SC2。k-HM-3对人甲状腺癌 SW-579裸小鼠移植瘤生长抑制试验结果表明， 与阴性对照 组相比， 多肽 36.7mg/kg组对人甲状腺癌 SW-579移植瘤的生长有显著性的抑制作用； 与阳 性对照 5-Fu组相比， 对实验动物的体重没有影响， 未见明显的毒副反应。 *Ρ〈0· 05 (和阴 性组比较有显著性差异） 实施例 10 mPEG-SC20k-HM-3对人胃癌 MGC803裸小鼠异种移植肿瘤生长抑制试验 取生长旺盛期的瘤组织剪切成 1.5mm3左右， 在无菌条件下， 接种于裸小鼠右侧皮下。 小鼠移植瘤用游标卡尺测量移植瘤直径， 待肿瘤生长至 100-200mm3后动物随机分组。 使用 测量瘤径的方法， 动态观察被试多肽抗肿瘤的效^。 肿瘤直径的测量次数为每 2天 1次， 每次测量同时还需称量鼠重。 实验组尾静脉注射多肽， 每三天 1 次， 阴性组同时给等量生 理盐水。 肿瘤体积计算公式：
TV=0.52 X aX b2 其中 a、 b分别表示长宽。 根据测量的结果计算出相对肿瘤体积。 抗肿瘤活性的评价指 标为相对肿瘤增殖率 T/C(%；)， 计算公式如下：
T/C(%)=TRTV/CRTV X 100%
TRTV: 治疗组 RTV; CRTV: 阴性对照组 RTV
表 5. mPEG-SC20k-HM-3对人胃癌 MGC803裸鼠异种移植肿瘤生长的抑制作用
起始数
剂量 起始体 终末体重
给药频率 抑瘤率
Figure imgf000015_0001
( mg/kg) 重 （g) (g) 阴性对
一 每天 次 22.32 12 22.15 0.723
照、
末动物数终
紫杉醇 10 两天 次 22.14 8 22.05 0.187 74.12% 恩度 2.5 每天 次 23.22 8 23.13 0.504 30.29%
HM-3 1.5 三天 次 22.52 8 22.37 0.214 70.40% mPEG-SC
20k-HM-3 36.7 次 22.67 8 22.24 0.192 73.42%* 高
mPEG-SC
20k-HM-3 18.75 次 22.76 8 22.32 0.218 69.86% mPEG-SC
-HM-3 9.38 三天 次 22.81 8 22.44 0.292 59.57% 低 结果： 见表 5和图 6， 紫杉醇组 10mg/kg， 对人胃癌 MGC803裸小鼠移植瘤的抑瘤率为 74.12%； 恩度组 2.5mg/kg， 对人胃癌 MGC803裸小鼠移植瘤的抑瘤率为 30.29%; HM-3组 1.5mg/kg， 对人胃癌 MGC803裸小鼠移植瘤的抑瘤率为 70.40%; 多肽高、 中、 低剂量组对 人胃癌 MGC803裸小鼠移植瘤的抑瘤率达 73.42%， 69.86%, 59.57%。 mPEG-SC20k-HM-3对人胃癌 MGC803裸小鼠移植瘤生长抑制试验结果表明， 与阴性对照组 相比，多肽 36.7mg/kg组对人胃癌 MGC803移植瘤的生长有显著性的抑制作用。 *P<0. (和 阴性组比较有显著性差异） 实施例 11 mPEG-SC2。k-HM-3对人胰腺癌 SW-1990裸小鼠异种移植肿瘤生长抑制试验 取生长旺盛期的瘤组织剪切成 1.5mm3左右， 在无菌条件下， 接种于裸小鼠右侧皮下。 小鼠移植瘤用游标卡尺测量移植瘤直径， 待肿瘤生长至 100-200mm3后动物随机分组。 使用 测量瘤径的方法， 动态观察被试多肽抗肿瘤的效应。 肿瘤直径的测量次数为每 2天 1次， 每次测量同时还需称量鼠重。 实验组尾静脉注射多肽， 每三天 1 次， 阴性组同时给等量生 理盐水。 肿瘤体积计算公式：
TV=0.52 X aX b2 其中 a、 b分别表示长宽。 根据测量的结果计算出相对肿瘤体积。 抗肿瘤活性的评价指 标为相对肿瘤增殖率 T/C(%；)， 计算公式如下：
T/C(%)=TRTV/CRTV X 100%
TRTV: 治疗组 RTV; CRTV: 阴性对照组 RTV
表 6. mPEG-SC20k-HM-3对人胰腺癌 SW-1990裸鼠异种移植肿瘤生长的抑制作用
抑瘤率
Figure imgf000016_0001
阴性对
一 5 一 眧 每天一次 22.30 12 22.18 12 1.38
5-Fu 10 每天一次 23.17 8 22.43 8 0.323 76.68%
2.5 每天一次 24.26 8 23.73 8 0.932 32.71%
HM-3 1.5 三天一次 23.40 8 23.10 8 0.582 57.94% mPEG-SC
20k-HM-3 36. 三天一次 24.63 8 23.32 8 0.446 67.76% 高
mPEG-SC
20k-HM-3 18.75 三天一次 23.33 8 23.17 8 0.491 64.55% 中
mPEG-SC
9.38 三天一次 23.34 8 22.67 8 0.687 50.40% 20k-HM-3 结果： 见表 6和图 7， 5-Fu组 10mg/kg， 对人胰腺癌 SW-1990裸小鼠移植瘤的抑瘤率 为 76.68%;恩度组 2.5mg/kg，对人胰腺癌 SW-1990裸小鼠移植瘤的抑瘤率为 32.71%; HM-3 组 1.5mg/kg， 对人胰腺癌 SW-1990裸小鼠移植瘤的抑瘤率为 57.94%; 多肽高、 中、 低剂量 组对人胰腺癌 SW-1990裸小鼠移植瘤的抑瘤率达 67.76%， 64.55%, 50.40%。
mPEG-SC2。k-HM-3对人胰腺癌 SW-1990裸小鼠移植瘤生长抑制试验结果表明， 与阴性对照 组相比，多肽 36.7mg/kg组对人胰腺癌 SW-1990移植瘤的生长有显著性的抑制作用。 *Ρ〈0· 05
(和阴性组比较有显著性差异）
实施例 12
mPEG-SC20k-HM-3对人肺癌 H460裸小鼠异种移植肿瘤生长抑制试验 取生长旺盛期的瘤组织剪切成 1.5mm3左右， 在无菌条件下， 接种于裸小鼠右侧皮下。 小鼠移植瘤用游标卡尺测量移植瘤直径， 待肿瘤生长至 100-200mm3后动物随机分组。 使用 测量瘤径的方法， 动态观察被试多肽抗肿瘤的效应。 肿瘤直径的测量次数为每 2天 1次， 每次测量同时还需称量鼠重。 实验组尾静脉注射多肽， 每三天 1 次， 阴性组同时给等量生 理盐水。 肿瘤体积计算公式：
TV=0.52 X a X b2 其中 a、 b分别表示长宽。 根据测量的结果计算出相对肿瘤体积。 抗肿瘤活性的评价指 标为相对肿瘤增殖率 T/C(%；)， 计算公式如下：
T/C(%)=TRTV/CRTV X 100%
TRTV: 治疗组 RTV; CRTV: 阴性对照组 RTV
表 7. mPEG-SC20k-HM-3对人肺癌 H460裸鼠异种移植肿瘤生长的抑制作用
抑瘤率
Figure imgf000017_0001
i 对 ― 每天一次 21.36 12 21.21 12 0.794 ― 八1 *、
紫杉醇 10 两天一次 22.34 8 19.58 8 0.248 68.77% 恩度 2.5 每天一次 21.08 8 20.86 8 0.546 31.20% HM-3 1.5 三天一次 21.33 8 21.11 8 0.274 65.42% mPEG-SC
-HM-3 36.7 三天一次 21.47 8 20.79 8 0.266 66.45%* mPEG-SC
-HM-3 18.75 三天一次 22.38 8 22.16 8 0.354 55.37% 中
mPEG-SC
-HM-3 9.38 三天一次 21.28 8 21.06 8 0.363 54.28% 低 结果： 见表 7和图 8， 紫杉醇组 10mg/kg， 对人肺癌 H460裸小鼠移植瘤的抑瘤率为 68.77%； 恩度组 2.5mg/kg， 对人肺癌 H460 裸小鼠移植瘤的抑瘤率为 31.20%; HM-3 组 1.5mg/kg， 对人肺癌 H460裸小鼠移植瘤的抑瘤率为 65.42%; 多肽高、 中、低剂量组对人肺 癌 H460裸小鼠移植瘤的抑瘤率达 66.45%， 55.37%, 54.28%。 mPEG-SC20k-HM-3对人肺癌 H460裸小鼠移植瘤生长抑制试验结果表明， 与阴性对照组相比， 多肽 36.7mg/kg组对人肺癌 H460移植瘤的生长有显著性的抑制作用。 *Ρ<0·05 (和阴性组比 较有显著性差异）
实施例 13 mPEG-SC20k-HM-3对人乳腺癌 MDA-MB-231裸小鼠异种移植肿瘤生长抑制试验 取生长旺盛期的瘤组织剪切成 1.5mm3左右， 在无菌条件下， 接种于裸小鼠右侧皮下。 小鼠移植瘤用游标卡尺测量移植瘤直径， 待肿瘤生长至 100-200mm3后动物随机分组。 使用 测量瘤径的方法， 动态观察被试多肽抗肿瘤的效应。 肿瘤直径的测量次数为每 2天 1次， 每次测量同时还需称量鼠重。 实验组尾静脉注射多肽， 每天 1 次， 阴性组同时给等量生理 盐水。 肿瘤体积计算公式：
TV=0.52 X aX b2 其中 a、 b分别表示长宽。 根据测量的结果计算出相对肿瘤体积。 抗肿瘤活性的评价指 标为相对肿瘤增殖率 T/C(%；)， 计算公式如下：
T/C(%)=TRTV/CRTV X 100%
TRTV: 治疗组 RTV; CRTV: 阴性对照组 RTV 表 8. mPEG-SC20k-HM-3对人乳腺癌 MDA-MB-231裸鼠异种移植肿瘤生长的抑制作用 抑瘤率
Figure imgf000019_0001
阴性对
一 每天 次 22.32 10 22.69 1.243 ― 照、
紫杉醇 10 两天 次 23.90 8 23.32 0.335 73.05% 恩度 2.5 每天 次 24.65 8 23.03 0.801 35.57%
HM-3 1.5 三天 次 23.43 8 23.54 0.533 57. 14% mPEG-SC
36.7 次 22.38 8 22.73 0.459 63.05%* 18.75 次 23.66 8 22.34 0.508 59. 11% 9.38 次 23.73 8 23.49 0.609 51.01% 结果： 见表 8和图 9， 紫杉醇组 10mg/kg， 对人乳腺癌 MDA-MB-23 1裸小鼠移植瘤的 抑瘤率为 73.05% ; 恩度组 2.5mg/kg， 对人乳腺癌 MDA-MB-23 1裸小鼠移植瘤的抑瘤率为 35.57%； HM-3组 1.5mg/kg， 对人乳腺癌 MDA-MB-23 1裸小鼠移植瘤的抑瘤率为 57. 14% ; 多肽高、中、低剂量组对人乳腺癌 MDA-MB-23 1裸小鼠移植瘤的抑瘤率达 63.05%， 59. 11% , 51.01%。 mPEG-SC20k-HM-3对人乳腺癌 MDA-MB-23 1裸小鼠移植瘤生长抑制试验结果表明， 与阴性 对照组相比， 多肽 36.7mg/kg组对人乳腺癌 MDA-MB-23 1移植瘤的生长有显著性的抑制作 用。 *P<0.05 (和阴性组比较有显著性差异）
实施例 14 mPEG-SC20k-HM-3对人胆囊癌 GBC-SD裸小鼠异种移植肿瘤生长抑制试验 取对数生长期的人胆囊癌 GBC-SD细胞株，在无菌条件下后制备成 5 x l 07/ml细胞悬液， 以 0. 1 ml接种于裸小鼠右侧腋窝皮下。裸小鼠移植瘤用游标卡尺测量移植瘤直径，待肿瘤生 长至 100-200mm3后将动物随机分组。使用测量瘤径的方法，动态观察被试物抗肿瘤的效应。 肿瘤直径的测量次数为每 2天测 1次。 给药方式均采用尾静脉注射。 阴性对照组注射等量 生理盐水， 每天 1次； 紫杉醇组 10mg/kg， 每周给药 1次； 恩度组 2.5mg/kg， 每天给药 1 6， 3 , 1.5mg/kg， 每天给药 1次。 给药结束后， 小鼠处死， 手术 享度
剥取瘤块称重。 表 9. mPEG-SC20k-HM-3对人胆囊癌 GBC-SD裸鼠异种移植肿瘤生长的抑制作用
Figure imgf000020_0001
10 两天一次 20.52 20.43 8 0.232 78. 13%
2.5 每天一次 20.68 8 20.45 8 0.728 3 1.39%
HM-3 1.5 三天一次 21. 12 8 20.92 8 0.450 57.59% mPEG-
SC2ok-
36.7 三天一次 20.46 20.07 8 0.409 61.45%*
HM-3
高
mPEG-
SC2ok-
18.75 三天一次 21.32 8 20.56 8 0.524 50.59%
HM-3
中
mPEG-
SC2ok-
9.38 三天一次 21.05 8 20.58 8 0.633 40.32%
HM-3
低 结果： 见表 9和图 10， 紫杉醇组 10mg/kg， 对人胆囊癌 GBC-SD裸小鼠移植瘤的抑瘤 率为 78. 13% ; 恩度组 2.5mg/kg， 对人胆囊癌 GBC-SD裸小鼠抑制瘤的抑瘤率为 3 1.39% ; HM-3组 1.5mg/kg， 对人胆囊癌 GBC-SD裸小鼠移植瘤的抑瘤率为 57.59% ; 多肽高、 中、 低剂量组对人胆囊癌 GBC-SD裸小鼠移植瘤的抑瘤率为 61.45%， 50.59% , 40.32%。 mPEG-SC20k-HM-3多肽对人胆囊癌 GBC-SD小鼠移植瘤的抑瘤率的试验结果表明，与阴性对 照组相比，多肽 36.7mg/kg组对人胆囊癌 GBC-SD移植瘤的生长有显著性抑制作用。 *P<0.05
(和阴性组比较有显著性差异）
实施例 15 mPEG-SC2。k-HM-3对人肾癌 A498裸小鼠异种移植肿瘤生长抑制试验 取对数生长期的人肾癌 A498细胞株， 在无菌条件下后制备成 5 x l 07/ml细胞悬液， 以 0. 1ml接种于裸小鼠右侧腋窝皮下。 裸小鼠移植瘤用游标卡尺测量移植瘤直径， 待肿瘤生长 至 100-200mm3后将动物随机分组。 使用测量瘤径的方法， 动态观察被试物抗肿瘤的效应。 肿瘤直径的测量次数为每 2天测 1次。 给药方式均采用尾静脉注射。 阴性对照组注射等量 生理盐水， 每天 1次； 紫杉醇组 10mg/kg， 每周给药 1次； 恩度组 2.5mg/kg， 每天给药 1 次； 多肽高中低组分别以 6， 3 , 1.5mg/kg， 每天给药 1次。 给药结束后， 小鼠处死， 手术 剥取瘤块称重。 表 10. mPEG-SC20k-HM-3对人肾癌 A498裸鼠异种移植肿瘤生长的抑制作用 重
瘤重 ( g ) 抑瘤率
Figure imgf000021_0001
阴性对
一 每天 次 21.38 10 21. 11 0.944 ― 照、
紫杉醇 10 两天 次 21.23 6 21. 15 0.242 74.32% 恩度 2.5 每天 次 21.43 6 21.66 0.656 30.51%
HM-3 1.5 三天 次 20.47 8 20.88 0.427 54.77% mPEG-SC
20k-HM-3 36.7 次 20.00 6 20.23 末动物数终 0.424 55. 11%* 高
mPEG-SC
20k-HM-3 18.75 次 21.34 6 20.48 0.501 46.95% 中
mPEG-SC
20k-HM-3 9.38 次 20.35 6 20.76 0.576 39.00% 低 结果： 见表 10和图 11， 紫杉醇组 10mg/kg， 对人肾癌 A498裸小鼠移植瘤的抑瘤率为 84.32%； 恩度组 2.5mg/kg， 对人肾癌 A498 裸小鼠移植瘤的抑瘤率为 30.51% ; HM-3 组 1.5mg/kg， 对人肾癌 A498裸小鼠移植瘤的抑瘤率为 54.77% ; 多肽高、 中、低剂量组对人肾 癌 A498裸小鼠移植瘤的抑瘤率达 55. 11%， 46.95% , 39.00%。
mPEG-SC20k-HM-3对人肾癌 A498裸小鼠移植瘤生长抑制试验结果表明，与阴性对照组相比， 多肽 36.7mg/kg组对人肾癌 A498移植瘤的生长有显著性的抑制作用。 *Ρ〈0· 05 (和阴性组 比较有显著性差异）
实施例 16 mPEG-SC20k-HM-3对人结肠癌 HT-29裸小鼠异种移植肿瘤生长的抑制试验 取对数生长期的人结肠癌 HT-29细胞株， 在无菌条件下后制备成 5xl07/ml细胞悬液， 以 0.1ml接种于裸小鼠右侧腋窝皮下。裸小鼠移植瘤用游标卡尺测量移植瘤直径，待肿瘤生 长至 100-200mm3后将动物随机分组。使用测量瘤径的方法，动态观察被试物抗肿瘤的效应。 肿瘤直径的测量次数为每 2天测 1次。 给药方式均采用尾静脉注射。 阴性对照组注射等量 生理盐水， 每天 1次； 紫杉醇组 10mg/kg， 每周给药 1次； 恩度组 2.5mg/kg， 每天给药 1 次； 多肽高中低组分别以 6， 3, 1.5mg/kg， 每天给药 1次。 给药结束后， 小鼠处死， 手术 剥取瘤块称重。
表 11 mPEG-SC20k-HM-3对人结肠癌 HT-29裸鼠异种移植肿瘤生长的抑制作用 剂
起始体 终末体重
给药频率 抑瘤率 (<¼)
Figure imgf000022_0001
(mg/k 重 （g) (g)
起动数
g) 始物
阴性对照 一 每天— -次 23.94 12 23.67 12 1.120 一 紫杉醇 10 两天一 -次 23.43 8 22.84 6 0.346 69.11% 恩度 2.5 每天— -次 23.23 8 22.81 8 0.745 33.48%
HM-3 1.5 三天一 -次 24.11 12 23.15 12 0.531 52.59% 末动物数终
mPEG-SC20k-
36.7 三天一 -次 23.45 8 22.68 8 0.493 55.98%* HM-3高
mPEG-SC20k-
18.75 三天一 -次 24.61 8 25.36 8 0.611 45.45% HM-3中
mPEG-SC20k-
9.38 三天一 -次 24.29 8 23.17 8 0.705 37.05% HM-3低 结果： 见表 11和图 12， 紫杉醇组 10mg/kg， 对人结肠癌 HT-29裸小鼠移植瘤的抑瘤率 为 69.11%; 恩度组 2.5mg/kg， 对人结肠癌 HT-29裸小鼠移植瘤的抑瘤率为 33.48%; HM-3 组 1.5mg/kg， 对人结肠癌 HT-29裸小鼠移植瘤的抑瘤率为 52.59%; 多肽高、 中、 低剂量组 对人结肠癌 HT-29裸小鼠移植瘤的抑瘤率为 55.98%， 45.45%, 37.05% 因此， mPEG-SC2。k-HM-3对人结肠癌 HT-29裸小鼠移植瘤生长抑制试验结果表明， 与阴性 对照组相比， 多肽 36.7mg/kg 组对人结肠癌 HT-29 移植瘤的生长有显著性的抑制作用。
*P<0.05 (和阴性组比较有显著性差异）
实施例 17 mPEG-SC2。k-HM-3对人卵巢癌 SK-OV-3裸小鼠异种移植肿瘤生长的抑制试验 取对数生长期的人卵巢癌 SK-OV-3细胞株，在无菌条件下后制备成 5xl07/ml细胞悬液， 以 0. 1 ml接种于裸小鼠右侧腋窝皮下。裸小鼠移植瘤用游标卡尺测量移植瘤直径，待肿瘤生 长至 100-200mm3后将动物随机分组。使用测量瘤径的方法，动态观察被试物抗肿瘤的效应。 肿瘤直径的测量次数为每 2天测 1次。 给药方式均采用尾静脉注射。 阴性对照组注射等量 生理盐水， 每天 1次； 顺铂组 10mg/kg， 每周给药 1次； 恩度组 2.5mg/kg， 每天给药 1次； 多肽高中低组分别以 6， 3， 1.5mg/kg， 每天给药 1次。 给药结束后， 小鼠处死， 手术剥取 瘤块称重。 mPEG-SC2。k-HM-3对人卵巢癌 SK-OV-3裸鼠异种移植肿瘤生长的抑制作用
Figure imgf000023_0001
顺铂 10 每周两次 22.43 8 22. 19 6 0.302 76. 13% 恩度 2.5 每天一次 22.94 8 22.74 8 0.860 3 1.98%
HM-3 1.5 三天一次 22.90 8 22.85 8 0.639 49.49% mPEG-SC20k-
36.7 三天一次 23.53 8 22.35 8 0.627 50.40%* HM-3高
mPEG-SC20k-
18.75 三天一次 23.66 8 22.29 8 0.700 44.62% HM-3中
mPEG-SC20k-
9.38 三天一次 22.89 8 22.74 8 0.730 42.33% HM-3低 结果： 见表 12和图 13， 顺铂组 10mg/kg， 对人卵巢癌 SK-OV-3裸小鼠移植瘤的抑瘤率 为 76. 13% ;恩度组 2.5mg/kg，对人卵巢癌 SK-OV-3裸小鼠移植瘤的抑瘤率为 3 1.98% ; HM-3 组 1.5mg/kg， 对人卵巢癌 SK-OV-3裸小鼠移植瘤的抑瘤率为 49.49% ; 多肽高、 中、 低剂量 组对人卵巢癌 SK-OV-3裸小鼠移植瘤的抑瘤率为 50.40%， 44.62% , 42.33%。 因此， 整合素阻断剂多肽对人卵巢癌 SK-OV-3裸小鼠移植瘤生长抑制试验结果表明， 与阴性对照组相比， 多肽 36.7mg/kg组对人卵巢癌 SK-OV-3移植瘤的生长有显著性的抑制 作用。 *P<0.05 (和阴性组比较有显著性差异） 实施例 18 mPEG-SC20k-HM-3对人子宫内膜癌 HHUA裸小鼠异种移植肿瘤生长的抑制试验 取对数生长期的人子宫内膜癌 HHUA细胞株，在无菌条件下后制备成 5 x l 07/ml细胞悬 液， 以 0. 1 ml接种于裸小鼠右侧腋窝皮下。裸小鼠移植瘤用游标卡尺测量移植瘤直径， 待肿 瘤生长至 100-200mm3后将动物随机分组。 使用测量瘤径的方法， 动态观察被试物抗肿瘤的 效应。 肿瘤直径的测量次数为每 2天测 1次。 给药方式均采用尾静脉注射。 阴性对照组注 射等量生理盐水， 每天 1次； 紫杉醇组 10mg/kg， 每周给药 1次； 恩度组 2.5mg/kg， 每天给 药 1次； 多肽高中低组分别以 6， 3 , 1.5mg/kg， 每天给药 1次。 给药结束后， 小鼠处死， 手术剥取瘤块称重。 mPEG-SC20k-HM-3对人子宫内膜癌 HHUA裸鼠异种移植肿瘤生长的抑制作用 剂
華:
起始体 终末体 抑瘤率 组别 给药频率
( mg/ 重 （g ) 重 （g ) ( g ) (%) kg )
阴性对照 一 每天一次 24.51 12 23.03 12 1.208 一 紫杉醇 10 每周两次 23.43 ^ 8 ^ 22. 19 6 0.295 75.55% 恩度 2.5 每天一次 22.98 8 22.74 8 0.785 34.98%
HM-3 1.5 三天一次 22.94 8 22.85 8 0.550 54.49% mPEG-SC20k-
36.7 三天一次 23.98 8 22.39 8 0.453 62.47%* HM-3高
mPEG-SC20k-
18.75 三天一次 23.76 8 22.37 8 0.560 53.65% HM-3中 ¾·
mPEG-SC20k-
9.38 三天一次 22.89 8 22.66 8 0.587 51.38% HM-3低 结果： 见表 13和图 14， 紫杉醇组 10mg/kg， 对人子宫内膜癌 HHUA裸小鼠移植瘤的 抑瘤率为 75.55% ; 恩度组 2.5mg/kg， 对人子宫内膜癌 HHUA裸小鼠移植瘤的抑瘤率为 34.98%； HM-3组 1.5mg/kg， 对人子宫内膜癌 HHUA裸小鼠移植瘤的抑瘤率为 54.49% ; 多 肽高、 中、 低剂量组对人子宫内膜癌 HHUA裸小鼠移植瘤的抑瘤率为 62.47%， 53.65% , 51.38%。 . 因此， mPEG-SC2。k-HM-3对人子宫内膜癌 HHUA裸小鼠移植瘤生长抑制试验结果表明， 与 阴性对照组相比， 多肽 36.7mg/kg组对人子宫内膜癌 HHUA移植瘤的生长有显著性的抑制 作用。 *P<0.05 (和阴性组比较有显著性差异）
实施例 19 mPEG-SC20k-HM-3对人宫颈癌 HeLa裸小鼠异种移植肿瘤生长的抑制试验 取对数生长期的人宫颈癌 HeLa细胞株， 在无菌条件下后制备成 5 >< 107/ml细胞悬液， 以 0. 1 ml接种于裸小鼠右侧腋窝皮下。裸小鼠移植瘤用游标卡尺测量移植瘤直径，待肿瘤生 长至 100-200mm3后将动物随机分组。使用测量瘤径的方法，动态观察被试物抗肿瘤的效应。 肿瘤直径的测量次数为每 2天测 1次。 给药方式均采用尾静脉注射。 阴性对照组注射等量 生理盐水， 每天 1次； 紫杉醇组 10mg/kg， 每周给药 1次； 恩度组 2.5mg/kg， 每天给药 1 次； 多肽高中低组分别以 6， 3 , 1.5mg/kg， 每天给药 1次。 给药结束后， 小鼠处死， 手术 剥取瘤块称重。
表 14 mPEG-SC2。k-HM-3.对人宫颈癌 HeLa裸鼠异种移植肿瘤生长的抑制作用
剂
起始体 终末体重 抑瘤率 给药频率
重 （g ) ( g ) (%)
/kg )
起动
阴性对照 一 每天一次 23. 11 8始物 23.35 8 1.236 一 紫杉醇 10 两天一次 24.36 6 23.47 6 0.428 65.37% 恩度 2.5 每天一次 23.54 6 23.28 6 0.796 35.57%
HM-3 1.5 三天一次 24. 11 6 23.86 6 0.528 57.25% mPEG-SC20k-
36.7 三天一次 23.76 6 23.05 末动物数终 6 0.222 82.07%* HM-3高
mPEG-SC20k-
18.75 三天一次 24.80 6 24.43 6 0.320 74. 11% HM-3中
mPEG-SC20k-
9.38 三天一次 24. 10 6 23.85 6 0.453 63.32% HM-3低 结果： 见表 14和图 15、 16， 紫杉醇组 10mg/kg， 对人宫颈癌 HeLa裸小鼠移植瘤的抑 瘤率为 65.37% ;恩度组 2.5mg/kg，对人宫颈癌 HeLa裸小鼠移植瘤的抑瘤率为 35.57% ; HM-3 组 1.5mg/kg， 对人宫颈癌 HeLa裸小鼠移植瘤的抑瘤率为 57.25% ; 多肽高、 中、 低剂量组 对人宫颈癌 HeLa裸小鼠移植瘤的抑瘤率为 82.07%， 74. 11% , 63.32%。 . 因此， mPEG-SC2。k-HM-3对人宫颈癌 HeLa裸小鼠移植瘤生长抑制试验结果表明， 与阴性 对照组相比， 多肽 36.7mg/kg 组对人宫颈癌 HeLa 移植瘤的生长有显著性的抑制作用。
*P<0.05 (和阴性组比较有显著性差异）
实施例 20 mPEG-SC20k-HM-3对人前列腺癌 DU- 145裸小鼠异种移植肿瘤生长的抑制试验 取对数生长期的人前列腺癌 DU- 145细胞株， 在无菌条件下后制备成 5 x l 07/ml细胞悬 液， 以 0. 1 ml接种于裸小鼠右侧腋窝皮下。裸小鼠移植瘤用游标卡尺测量移植瘤直径， 待肿 瘤生长至 100-200mm3后将动物随机分组。 使用测量瘤径的方法， 动态观察被试物抗肿瘤的 效应。 肿瘤直径的测量次数为每 2天测 1次。 给药方式均采用尾静脉注射。 阴性对照组注 射等量生理盐水， 每天 1次； 顺铂组 10mg/kg， 每周给药 1次； 恩度组 2.5mg/kg， 每天给药 1次； 多肽高中低组分别以 6， 3， 1.5mg/kg， 每天给药 1次。 给药结束后， 小鼠处死， 手术 剥取瘤块称重。
表 15. mPEG-SC20k-HM-3对人前列腺癌 DU-145裸鼠异种移植肿瘤生长的抑制作用 起始体 终末体重 抑瘤率 给药频率
( mg/ 重 （g ) ( g ) (%) kg ) 阴性对照 一 每天一次 24. 12 12 23.65 12 1.854 一
起动
顺铂 10 每周两次 24.57 8始物 23.81 6 0.545 70.60% 恩度 2.5 每天一次 23.08 8 23.25 8 1.274 3 1.25%
HM-3 1.5 三天一次 24.77 8 23.64 8 0.809 56.36% mPEG-SC20k-
36.7 三天一次 23.26 8 23.08 8 0.417 77.46%* HM-3高
mPEG-SC20k- 末动物数终
18.75 三天一次 24.38 8 23.61 8 0.603 67.48% HM-3中
mPEG-SC20k-
9.38 三天一次 24.41 8 23.37 8 0.781 57.87% HM-3低 结果： 见表 15和图 17， 顺铂组 10mg/kg， 对人前列腺癌 DU- 145裸小鼠移植瘤的抑瘤 率为 70.60% ; 恩度组 2.5mg/kg， 对人前列腺癌 DU- 145裸小鼠移植瘤的抑瘤率为 3 1.25% ; HM-3组 1.5mg/kg， 对人前列腺癌 DU- 145裸小鼠移植瘤的抑瘤率为 56.36% ; 多肽高、 中、 低剂量组对人前列腺癌 DU- 145裸小鼠移植瘤的抑瘤率为 77.46%， 67.48% , 57.87%。 因此， mPEG-SC2。k-HM-3对人前列腺癌 DU- 145裸小鼠移植瘤生长抑制试验结果表明， 与 阴性对照组相比， 多肽 36.7mg/kg组对人前列腺癌 DU- 145移植瘤的生长有显著性的抑制作 用。 *P<0.05 (和阴性组比较有显著性差异）
实施例 21 mPEG-SC20k-HM-3对人膀胱癌 HT 1376裸小鼠异种移植肿瘤生长的抑制试验 取对数生长期的人膀胱癌 HT 1376细胞株， 在无菌条件下后制备成 5 x l 07/ml细胞悬液， 以 0. 1 ml接种于裸小鼠右侧腋窝皮下。裸小鼠移植瘤用游标卡尺测量移植瘤直径，待肿瘤生 长至 100-200mm3后将动物随机分组。使用测量瘤径的方法，动态观察被试物抗肿瘤的效应。 肿瘤直径的测量次数为每 2天测 1次。 给药方式均米用尾静脉注射。 阴性对照组注射等量 生理盐水， 每天 1次； 紫杉醇组 10mg/kg， 每周给药 1次； 恩度组 2.5mg/kg， 每天给药 1 次； 多肽高中低组分别以 6， 3 , 1.5mg/kg， 每天给药 1次。 给药结束后， 小鼠处死， 手术 剥取瘤块称重。
表 16. G-SC20k-HM-3对人膀胱癌 HT1376裸鼠异种移植肿瘤生长的抑制作用
Figure imgf000027_0001
)
阴性对照 一 每天— -次 21.71 10 21.32 10 1.018 一 紫杉醇 10 两天一 -次 21.39 6 21.65 5 0.327 67.88% 恩度 2.5 每天— -次 21.52 21.36 6 0.698 3 1.43%
HM-3 1.5 三天一 -次 22.84 6 22.30 6 0.504 50.49% mPEG-SC20k-
36.7 三天一 -次 22.27 6 21.59 6 0.372 63.42%* HM-3高
mPEG-SC20k-
18.75 三天一 -次 21.58 6 21.49 6 0.466 54.24% HM-3中
mPEG-SC20k-
9.38 三天一 -次 22.76 6 22.23 6 0.546 46.39% HM-3低 结果： 见表 16和图 18、 19， 紫杉醇组 10mg/kg， 对人膀胱癌 HT 1376裸小鼠移植瘤的 抑瘤率为 67.88 ; 恩度组 2.5mg/kg， 对人膀胱癌 HT 1376裸小鼠移植瘤的抑瘤率为 3 1.43% ; HM-3组 1.5mg/kg， 对人膀胱癌 HT 1376裸小鼠移植瘤的抑瘤率为 50.49% ; 多肽高、 中、 低剂量组对人膀胱癌 HT 1376裸小鼠移植瘤的抑瘤率为 63.42%， 54.24% , 46.39%。
因此，整合素阻断剂多肽对人膀胱癌 HT 1376裸小鼠移植瘤生长抑制试验结果表明， 与 阴性对照组相比，多肽 36.7mg/kg组对人膀胱癌 HT 1376移植瘤的生长有显著性的抑制作用。
*P<0.05 (和阴性组比较有显著性差异）
实施例 22 mPEG-SC20k-HM-3对人睾丸癌 5637裸小鼠异种移植肿瘤生长的抑制试验 取对数生长期的人睾丸癌 5637细胞株， 在无菌条件下后制备成 5 x l 07/ml细胞悬液， 以 0. 1ml接种于裸小鼠右侧腋窝皮下。 裸小鼠移植瘤用游标卡尺测量移植瘤直径， 待肿瘤生长 至 100-200mm3后将动物随机分组。 使用测量瘤径的方法， 动态观察被试物抗肿瘤的效应。 肿瘤直径的测量次数为每 2天测 1次。 给药方式均采用尾静脉注射。 阴性对照组注射等量 生理盐水， 每天 1次； 顺铂组 10mg/kg， 每周给药 1次； 恩度组 2.5mg/kg， 每天给药 1次; 多肽高中低组分别以 6， 3， 1.5mg/kg， 每天给药 1次。 给药结束后， 小鼠处死， 手术剥取 瘤块称重。 表 17. mPEG-SC20k-HM-3对人睾丸癌 5637裸鼠异种移植肿瘤生长的抑制用 起始体 终末体重
给药频率 抑瘤率 (<¼)
Figure imgf000028_0001
( mg/ 重 （g ) ( g )
kg ) 阴性对照 一 每天一次 24.08 10 23.91 10 2.015 一 顺铂 10 每周两次 24.73 6 24.46 6 0.590 70.74% 恩度 2.5 每天一次 25.07 起动 6 24.89 6 1.410 30.02% 始物
HM-3 1.5 三天一次 25.66 6 25. 15 6 0.82 59.40% mPEG-SC20k-
36.7 三天一次 25.45 6 24. 18 6 0.802 60.20%* HM-3高
mPEG-SC20k-
18.75 三天一次 24.24 6 24. 19 6 0.981 51.32% HM-3中
mPEG-SC20k- 末动物数终
9.38 三天一次 25.74 6 25. 18 6 0.988 50.99% HM-3低 结果： 见表 17和图 20， 顺铂组 10mg/kg， 对人睾丸癌 5637裸小鼠移植瘤的抑瘤率为 70.74%； 恩度组 2.5mg/kg， 对人睾丸癌 5637裸小鼠移植瘤的抑瘤率为 30.02% ; HM-3组 1.5mg/kg， 对人睾丸癌 5637裸小鼠移植瘤的抑瘤率为 39.40% ; 多肽高、 中、 低剂量组对人 睾丸癌 5637裸小鼠移植瘤的抑瘤率为 60.20%， 51.32% , 50.99%。 因此， mPEG-SC2。k-HM-3对人睾丸癌 5637裸小鼠移植瘤生长抑制试验结果表明， 与阴性对 照组相比，多肽 36.7mg/kg组对人睾丸癌 5637移植瘤的生长有显著性的抑制作用。 *Ρ〈0· 05
(和阴性组比较有显著性差异）
实施例 25 mPEG-SC20k-HM-3对肉瘤 HT- 1080裸小鼠异种移植肿瘤生长的抑制试验 取对数生长期的肉瘤 HT- 1080细胞株， 在无菌条件下后制备成 5 x l 07/ml细胞悬液， 以 0. 1ml接种于裸小鼠右侧腋窝皮下。 裸小鼠移植瘤用游标卡尺测量移植瘤直径， 待肿瘤生长 至 100-200mm3后将动物随机分组。 使用测量瘤径的方法， 动态观察被试物抗肿瘤的效应。 肿瘤直径的测量次数为每 2天测 1次。 给药方式均采用尾静脉注射。 阴性对照组注射等量 生理盐水， 每天 1次； 环磷酰胺组 15mg/kg， 每周给药 1次； 多肽以 3mg/kg， 每天给药 1 次。 给药结束后， 小鼠处死， 手术剥取瘤块称重。
表 18. mPEG-SC20k-HM-3对肉瘤 HT-1080裸鼠异种移植肿瘤生长的抑制作用
剂
起始体 终末体重 抑瘤率 给药频率
重 （g) (g) (%) /kg) 阴性对照 一 每天一次 23.13 8 22.81 8 1.938 一 环磷酰胺 15 两天一次 24.09 8 23.85 8 0.501 74.15%
HM-3 1.5 三天一次 24.20 8 23.89 8 0.790 59.24% mPEG-SC20k-
36.7 三天一次 23.57 8 23.26 8 0.678 65.04%* HM-3 起动
始物
结果： 见表 18 和图 21， 环磷酰胺组 10mg/kg， 对肉瘤 HT-1080裸小鼠移植瘤的抑瘤率为 74.15%； HM-3组 1.5mg/kg， 对人肉瘤 HT-1080裸小鼠移植瘤的抑瘤率为 59.24%; 多肽组对肉瘤 HT-1080裸小鼠移植瘤的抑瘤率为 65.04%。 因此， mPEG-SC2。k-HM-3对肉瘤 HT-1080裸小鼠移植瘤生长抑制试验结末动物数终果表明， 与阴性对照组相 比， 多肽 36.7mg/kg组对 HT-1080移植瘤的生长有显著性的抑制作用。 *Ρ〈0·05 (和阴性组比较有 显著性差异）

Claims

权利 要 求 书 、 一种聚乙二醇修饰的整合素阻断剂 HM-3， 其中整合素阻断剂的序列为 mPEG-Ile-Val-Arg-Arg-Ala-Asp-Arg-Ala-Ala-Val-Pro-Gly-Gly- Gly-Gly -Arg-Gly-Asp, 其特征 在于所述的 mPEG-SC的分子量范围为 500-20000。
、 根据权利要求 1 所述的一种聚乙二醇修饰的整合素阻断剂 HM-3， 其特征在于所述的 mPEG-SC的分子量为 20000。
、 根据权利要求 1所述的一种聚乙二醇修饰的整合素阻断剂 HM-3在制备治疗肿瘤药物中的 应用， 其特征在于所述的肿瘤为起源于人的胃部、 皮肤、 头颈部、 甲状腺、 胰腺、 肺脏、 食管、 乳腺、 肾脏、 胆囊、 结肠 /直肠、 卵巢、 子宫、 子宫颈、 前列腺、 膀胱、 睾丸的原发 / 继发的癌或肉瘤。
PCT/CN2012/084788 2011-11-21 2012-11-17 一种聚乙二醇修饰的整合素阻断剂hm-3及其应用 WO2013075600A1 (zh)

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