Patent Application
20170100489
In accordance with the requirements of 37 C.F.R. §1.821(c), this application includes a Sequence Listing submitted on a compact disk in compliance with the requirements set forth in 37 C.F.R. §1.52(e). In accordance with the requirements set forth in 37 C.F.R. §1.52(e)(5), the contents of the Sequence Listing are hereby incorporated by reference.
The present invention involves the pharmaceutical field, including an integrin inhibitor, which has the capacities of inhibiting angiogenesis of tumors, binding integrin. This inhibitor is a kind of polypeptide, which was modified by polyethylene glycol and after modification, it can be used to treat tumors.
According to research, the growth of solid tumors relies on angiogenesis, which can not only provide nutrients and oxygen tumors need and excrete metabolites, but also the approach of distant metastasis. Therefore, blocking angiogenesis is a method that can prevent tumor growth and metastasis. And this theory stimulates extensive research on the pro-angiogenic molecules and anti-angiogenic molecules. Compared with the traditional medicine of tumors, the advantages of angiogenesis inhibitor are as follows: (1) Selectively affect vascular endothelial cells, and have a relatively lower systemic toxic side effect. (2) The target cells are vascular endothelial cells. Medicine is easy to reach and take effect. (3)
Due to no or rare vascular endothelial cell mutant occurring, it is not easy to produce drug resistance, and the patients can have a long-term medication. (4) Can be combined with chemotherapy methods and reduce the toxicity of the latter.
Currently, the integrin antagonists exploited internationally has entered phase II clinical experiment, whereas there have been no similar products entering Chinese market. So it is necessary to develop such drugs of our own intellectual property. Now several antagonists are introduced. One of them is integrin antagonist polypeptide, and its sequence is Ile-Val-Arg-Arg-Ala-Asp-Arg-Ala-Ala-Val-Pro-Gly-Gly-Gly-Gly-Arg-Gly-Asp (SEQ ID NO: 1), which contains integrin ligand sequences (Gly-Gly-Gly-Gly-Arg-Gly-Asp) (SEQ ID NO: 2) and angiogenesis inhibition sequence (Ile-Val-Arg-Arg-Ala-Asp-Arg-Ala-Ala-Val-Pro) (SEQ ID NO: 3). Integrin ligand sequences contain RGD sequences (Arg-Gly-Asp). Integrin antagonist polypeptide sequence can effectively bind integrin subtypes of tumor-specific expression and inhibit angiogenesis of tumors because of its angiogenesis inhibition sequence, eventually come to the results of preventing growth and metastasis of tumors. Previous research demonstrated that its targets are integrin ανβ3 and α5β1, and ανβ3 is the prime target. The polypeptide was testified to have better anti-tumor effect via repeated activity evaluation in vivo and vitro. It can significantly inhibit endothelial cell migration and tumor angiogenesis, thereby inhibit tumor growth. However, the half-life of the polypeptide is short. Clinically, this medicine is administered through intravenous infusion every day, so it brings the patients some pain.
According to report, it is a common method to solve the problems of continuous administration due to short half-time via modifying or alter molecular structure. Chemical modification is the most widely used. The chemical modifiers commonly used include polyethylene glycol (PEG), dextran, polyamino acids, polyanhydrides and so on. PEG has been approved as pharmaceutical auxiliary material and modifier by the U.S. Food and Drug Administration (FDA) because of the feature of non-toxicity, non-immunogenicity, and good water solubility. The molecular weight of protein drugs increases, while glomerular filtration rate decrease after PEG modification. PEG barrier protects protein from being hydrolyzed by protein proteolysis, meantime reduces the generation of neutralizing antibodies, which can extend the biological half-life of are protein drugs. Nowadays, there are a variety of protein drugs modified by PEG on sale. But PEG modification can also affect the biological activity of protein, and the degree of influence relates to modifier, modifying conditions and the nature of the protein itself. For specific proteins, its best modification is determined by proteins modified by PEG and its biological activity. Although the study of PEG started recently, it has attracted a lot of attention from researchers.
The invention made a further research for mPEG—SC-Ile-Val-Arg-Arg-Ala-Asp-Arg-Ala-Ala-Val-Pro-Gly-Gly-Gly-Gly-Arg-Gly-Asp (SEQ ID NO: 4), and discovered that it had therapeutic effect under the condition of decreasing the frequency of administration.
HM-3 is a kind of integrin inhibitor modified by polyethylene glycol and its sequence is mPEG-SC-Ile-Val-Arg-Arg-Ala-Asp-Arg-Ala-Ala-Val-Pro-Gly-Gly-Gly-Gly-Arg-Gly-Asp (SEQ ID NO: 4). And the feature of this inhibitor is the range of molecular weight of mPEG-SC is 500-20000.The feature of HM-3 mentioned above is its molecular weight of mPEG-SC is 20000 and its treatment of tumor originated from stomach, skin, head and neck, thyroid, pancreas, lung, esophagus, breast, kidney, gall bladder, colon/rectum, ovary, uterus, cervix, prostate, bladder, testicular primary/secondary cancer or sarcoma.
1. In order to extend the half-life of Ile-Val-Arg-Arg-Ala-Asp-Arg-Ala-Ala-Val-Pro-Gly-Gly-Gly-Gly-Arg-Gly-Asp (SEQ ID NO: 1), we used PEG of various molecular weights to modify this polypeptide and discovered that mPEG-SC2Qk-HM-3 had the role of extending half-time of HM-3, meantime, the feature of no impact of activity in vivo and vitro. A product modified by polypeptide is a novel molecule. It usually produces different effects compared with the molecule without modification. This invention made many studies about therapeutic effect of mPEG-SC20k-HM-3 on many tumors and discovered that mPEG-SC20k-HM-3 played a positive role in inhibiting growth of many tumors, and expanded its social and economic value.
2. According to study, sequence of Ile-Val-Arg-Arg-Ala-Asp-Arg-Ala-Ala-Val-Pro (SEQ ID NO: 3) had the function of inhibiting angiogenesis of tumors. The sequence of RGD was an important integrin ligand, so RGD-containing Gly-Gly-Gly-Gly-Arg-Gly-Asp (SEQ ID NO: 2) could specifically recognize integrin. The integrin antagonist polypeptide could bind Gly-Gly-Gly-Gly-Arg-Gly-Asp (SEQ ID NO: 2) sequence of RGD at C terminal of Ile-Val-Arg-Arg-Ala-Asp-Arg-Ala-Ala-Val-Pro (SEQ ID NO: 3) which had the function of inhibiting angiogenesis of tumors. Meantime, polyethylene glycol modification was made at the N terminal of the integrin antagonist polypeptide, and eventually formed the sequence: mPEG-SC20k-Ile-Val-Arg-Arg-Ala-Asp-Arg-Ala-Ala-Val-Pro-Gly-Gly-Gly-Gly-Arg-Gly-Asp (SEQ ID NO: 4), which contained PEG and 18 amino acid polypeptide. RGD sequence had the ability of integrin affinity and binding. Study demonstrated that the targets are integrin ανβ3 and α5β1, but the main target is ανβ3. RGD sequence contained angiogenesis inhibition sequence, eventually come to the results of preventing growth and metastasis of tumors. PEG is a kind of macromolecule polymer with unique physical and chemical properties. It has good biocompatibility and is non-toxic, non-antigenic. Main biological functions of protein or polypeptide drugs not only remain unchanged after undergoing PEG modification, but also can give protein or polypeptide drugs many excellent performances: (1) Increase stability, extend plasma half-life; (2) Reduce immunogenicity and antigenicity; (3) Reduce toxicity; (4) Reduce the possibility of degradation by hydrolytic enzymes and the rate of renal clearance; (5) Improve the distribution and dynamics of drug in the body. The target and anti-tumor activity of mPEG-SC20k polypeptide remain unchanged after modification, and extend half-time, reduce clearance and immunogenicity and antigenicity. Therefore, reduce the frequency of administration: from being administered once daily to once every 2-3 days. The results confirm that the integrin has advantages of significant anti-tumor effects, lower side effects, less dose and lower cost through many experiments, which demonstrated that the PEG modified integrin antagonist is scientific, reasonable, feasible and effective and can become treatment medicine of human anti-tumor. It can provide novel ideas and perspectives for future drug development, and expanded its social and economic value.
Half time of HM-3 before modification is 0.46 h, and the half time after modification by mPEG-SC20k is 20.13 h as indicated in the table below.
Some of the embodiments will be described in detail, with reference to the following figures, wherein like designations denote like members, wherein:
The method of synthesizing solid-state of Ile-Val-Arg-Arg-Ala-Asp-Arg-Ala-Ala-Val-Pro-Gly-Gly-Gly-Gly-Arg-Gly-Asp (SEQ ID NO: 1):Fmoc-Ile-wang resin or Fmoc-Ile-CTC resin is regarded as a raw material and then protect amino acids. Dipeptides to eighteen peptide are sequentially connected, then washed thoroughly, followed by peptide cut and post-management to obtain a crude product of HM-3. The crude product was purified. At first, it is dissolved, and then purified twice by preparative HPLC, and finally lyophilized to give a pure product. Specific steps are as follows:
Weigh Fmoc-Ile-Wang resin 14.7 g, and then pour it into a glass sand core reaction column of 1 L volume. The CH2CI2 147 ml was added to make resin full expansion.
Uncapping: After the solution of uncapping 25 ml with hexahydropyridine/DMF is sealed, keep it in the shaker for 5 minutes under the temperature of room temperature. Then the uncapping solution is drained, and washed once again with DMF in the middle and then add 20% uncapping solution 25 ml with a reaction of 15 minutes;
Washing: Drained the uncapping solution, and then is washed six times with the DMF resin, drained, and then take 20 resin into a small test tube, add test reagent, and heated under the temperature of 115° C. for 3 minutes.
Condensating: Weigh protective amino acids and HOBt 2.025 g, then be dissolved in 15 ml DMF and 2.33 ml DIC, then pour it into the reactor lasting about 1.5 hours, the temperature is controlled at about 34° C.
Washing: Drain the reaction solution, and wash the resin with DMF three times, drain again, and then put 10-20 resin particles into a small test tube, add test toner and heat it at 115° C. 3-5 min.
Cutting: Put the drained resin into round bottom flask of 500 mL volume, and then add 90% cleavage solution of 300 mL (phenol: thioanisole: EDT=90:3:3:2:2), and seal for 2 hours. Separate polypeptide from resin with a frit funnel
Postconditioning: At first add anhydrous ether to cleavage solution to precipitate the polypeptide, then centrifuge, then the supernatant was discarded, and then polypeptide is washed with anhydrous ether six times. Finally, drain it to gain crude peptide 9.5 g
Dissolving: Weigh ID-18 crude product exactly, then add purified water to the appropriate configuration of 10 g/1 solution. Ultrasonic stirs until a clear solution without particulate comes up.
Filtrating: The ID-18 solution is filtrated with 0.45 um membrane of sand core filter.
Balancing: Configure the solution of 5% acetonitrile and 95% aqueous trifluoroacetic acid. Rinse 10 min at a flow rate 80 ml/min.
Loading: Load with the infusion pump, flow rate 80 ml/min. And collect baseline and solution which is greater in the UV wavelength absorption of 220 nm than 200 mv to detect whether there are samples out.
Eluting: Gradient of Elution
Collect solution which is stronger in the UV wavelength absorption of 220 nm than 200 mv UV. The purity of greater than 95% is regarded as a peak and ready to do a secondary separation and purification.
Balancing: Configure the solution of 5% acetonitrile +95% aqueous acetic acid. Rinse 10 min at a flow rate 80 ml/min.
Loading: Load the primary peak with infusion pump after evaporating the organic solvent. And collect baseline and solution which is greater in the UV wavelength absorption of 220 nm than 200 mv to detect whether there are samples out.
Eluting: Gradient of Elution
Collect the solution which is stronger in the UV wavelength absorption of 220 nm than 200 mv. The purity of greater than 99% is regarded as qualification. Concentrating, filtering and freeze-drying: Concentrate the qualified solution with a rotary evaporator under 37° C. and remove residual solvent and water. Finally filter it with 0.22 um filter membrane and put the filtrate into the freeze-dried pan, then freeze and dry it with a freeze dryer to give pure product.
The reaction of mPEG-SC2OK and the HM-3
Weigh 2 g mPEG-SC20 k and 106.24 mg HM-3 (molar ratio 1.5:1) respectively. Both of them are placed in 40 ml-100 ml pH 5-8.5 PBS buffer solution at the conditions at 4° C. overnight and allow them to react. PEG-SC500-20000 can be connected as described in Example 2 to produce modified polypeptides
The sample after the reaction is purified through semi-preparative HPLC (HPLC, BIO-RAD) and purification conditions are as follows:
The collected products through HPLC are frozen in the cryogenic freezer at −70° C. overnight, then freeze and dry them through the freeze dryer until the products become white powder (30 h or so). Gain lyophilized product, weigh and record the weight of the products, and then save them in the −20° C. refrigerator and make identification.
The products are lyophilized and analyzed by analytical HPLC. The conditions of purity analysis are as follows:
Basic operation reference “Molecular Cloning (The second edition).” Concentration of stacking gel is 5%, and separating gel 10%, concentrated voltage 80 volts, separation voltage 120 volts. The bands of samples are stained with Bal2 first after electrophoresis especially for the portion containing PEG; Coomassie brilliant blue R250 as a Marker stains part of the protein. Place it into bleaching solution after staining to the effect of background transparent and then analyze the results through scanning The following takes mPEG20000 modified polypeptide integrin antagonist polypeptide (mPEG-SC20 k-HM-3.for example to make description. Integrin antagonist polypeptide described in example is the integrin antagonist polypeptide modified by mPEG20000.
SD rats were randomly divided into six groups with the same number for male and female. Take three groups were administered intravenously integrin antagonist polypeptide with a high dose of 52 mg/kg (equivalent to HM-3 4.2 mg/kg), an intermediate dose of 26 mg/kg (equivalent to HM-3 2.1 mg/kg), a low-dose of 13 mg/kg (equivalent to HM-3 1.05 mg/kg). The other 3 groups were injected HM-3 with a high dose of 4.2 mg/kg, an intermediate dose of 2.1 mg/kg, a low doses of 1.05 mg/kg. Collected whole blood 0.5 ml once from the orbital venous plexus after 0.5 h, 1 h, 2 h, 3 h, 6 h, 12 h, 24 h, 48 h, 72 h, 96 h, 108 h, 132 h of drug administration, and applied heparin to get the effect of anti-coagulation. 12000 rpm/2 min centrifuged plasma. Draw supernatant 200 μl and 80° C. preheated PBS (0.05 M pH7.4) buffer 600 μl and mixed. Bath in the 80° C. water for 30 min. Centrifuged 2 min at 12000 rpm, and collected the supernatant, and stored in the condition of −20° C. Dissolved at room temperature, measured integrin antagonist peptide plasma concentration by ELISA.
The Comparison of Pharmacokinetic Parameters of mPEG-SC20 k-HM-3 and HM-3 in SD Rats (t1/2βHalt-Time, CL the Rate of Plasma Clearance, AUD Area under the Curve, MRT the Average Residence Time)
As known from Table 1, compared with the HM-3, mPEG-SC20k-HM-3 has a significantly longer half-life and lower plasma clearance. The above experimental data have demonstrated PEG-modified protein can significantly improve the feature of pharmacokinetics of polypeptide drug in rats
BALB/c white mice were randomly divided into 2 groups, and the same number of male and female. 36 mg/kg mPEG-SC20k-HM-3 and 3.0 mg/kg HM-3 were administrated intravenously through tail vein, respectively, lasting 8 weeks, and collected blood from the orbital venous plexus once a week during 1-12 weeks. Centrifuged 2 min at 12000 rpm, collected the supernatant, and stored in the condition of −20° C. Dissolved at room temperature, 0.1 ml was used to measure antibody titers in serum by ELISA.
Group 1 The effective dose of HM-3 was 3.0 mg/kg. Administration: once a day, 6 rats (male, 3; female, 3)
Group 2 The effective dose of mPEG-SC20k-HM-3 was 36 mg/kg. Administration: once two days, 6 rats (male, 3; female, 3)
Picked the tumor tissue of vigorous growth and was cut into pieces of about 1.5 mm3. Under sterile conditions, the tumor tissue was inoculated subcutaneously on the right of nude mice. Tumor diameter was measured with a vernier caliper. The animals which had tumors with a size of 100-200 mm3 were randomly divided into groups. The effect of polypeptide on anti-tumor was dynamically observed through the method of measuring tumor diameter. The times of measuring tumor diameter were once 2 days, and meantime, weighed mice. The mice in experimental group were infused polypeptide through tail vein, once 3 days. The mice in the placebo group were injected saline with the same volume. Tumor volume formula:
TV=0.52×a×b2
a, b respectively represent the length and width. According to the result of measurement, calculate the relative tumor volume. Evaluation of antitumor activity is relative tumor proliferation rate T/C(%). The formula is as follows:
T/C(%)=TRTV/CRTV×100%
TRTV: Treatment Group RTV; CRTV: Negative Control Group RTV
Results: shown in table 2 and
The results of experiment of tumor growth inhibition induced by mPEG-SC20k-HM-3 on human esophageal cancer Ec109 xenograft tumors in nude mice demonstrated that compared with negative control group, mPEG-SC20K-HM-3 36.7 mg/kg played a significant inhibition role in growth of human esophageal cancer Ec109. *P<0.05 (there is significant difference compared with negative group).
Picked the tumor tissue of vigorous growth and was cut into pieces of about 1.5 mm3. Under sterile conditions, the tumor tissue was inoculated subcutaneously on the right of nude mice. Tumor diameter was measured with a vernier caliper. The animals which had tumors with a size of 100-200 mm3 were randomly divided into groups. The effect of polypeptide on anti-tumor was dynamically observed through the method of measuring tumor diameter. The times of measuring tumor diameter were once 2 days, and meantime, weighed mice. The mice in experimental group were infused polypeptide through tail vein, once 3 days. The mice in the placebo group were injected saline with the same volume. Tumor volume formula:
TV=0.52×a×b2
a, b respectively represent the length and width. According to the result of measurement, calculate the relative tumor volume. Evaluation of antitumor activity is relative tumor proliferation rate T/C(%). The formula is as follows: T/C(%)−TRTV/CRTV×100%
TRTV: Treatment Group RTV; CRTV: Negative Control Group RTV
Results: shown in table 3 and
The results of experiment of tumor growth inhibition induced by mPEG-SC20k-HM-3 on human nasopharyngeal cancer CNE xenograft tumors in nude mice demonstrated that compared with negative control group, mPEG-SC20K-HM-3 36.7 mg/kg played a significant inhibition role in growth of human nasopharyngeal cancer CNE; compared with positive cisplatin group, there was no significant influence on the weight of experimental mice, and no obvious toxic and side effects. *P<0.05 (there is significant difference compared with negative group)
Picked the tumor tissue of vigorous growth and was cut into pieces of about 1.5 mm3. Under sterile conditions, the tumor tissue was inoculated subcutaneously on the right of nude mice. Tumor diameter was measured with a vernier caliper. The animals which had tumors with a size of 100-200 mm3 were randomly divided into groups. The effect of polypeptide on anti-tumor was dynamically observed through the method of measuring tumor diameter. The times of measuring tumor diameter were once 2 days, and meantime, weighed mice. The mice in experimental group were infused polypeptide through tail vein, once 3 days. The mice in the placebo group were injected saline with the same volume. Tumor volume formula:
TV=0.52×a×b2
a, b respectively represent the length and width. According to the result of measurement, calculate the relative tumor volume. Evaluation of antitumor activity is relative tumor proliferation rate T/C(%). The formula is as follows: T/C(%)=TRTV/CRTV×100%
TRTV: Treatment Group RTV; CRTV: Negative Control Group RTV
Results: shown in table 4 and
The results of experiment of tumor growth inhibition induced by mPEG-SC20k-HM-3 on human thyroid cancer SW-579 xenograft tumors in nude mice demonstrated that compared with negative control group, mPEG-SC20K-HM-3 36.7 mg/kg played a significant inhibition role in growth of human thyroid cancer SW-579; compared with positive 5-Fu group, there was no significant influence on the weight of experimental mice, and no obvious toxic and side effects. *P<0.05 (there is significant difference compared with negative group).
Picked the tumor tissue of vigorous growth and was cut into pieces of about 1.5 mm3. Under sterile conditions, the tumor tissue was inoculated subcutaneously on the right of nude mice. Tumor diameter was measured with a vernier caliper. The animals which had tumors with a size of 100-200 mm3 were randomly divided into groups. The effect of polypeptide on anti-tumor was dynamically observed through the method of measuring tumor diameter. The times of measuring tumor diameter were once 2 days, and meantime, weighed mice. The mice in experimental group were infused polypeptide through tail vein, once 3 days. The mice in the placebo group were injected saline with the same volume. Tumor volume formula:
TV=0.52×a×b2
a, b respectively represent the length and width. According to the result of measurement, calculate the relative tumor volume. Evaluation of antitumor activity is relative tumor proliferation rate T/C(%). The formula is as follows: T/C(%)=TRTV/CRTV×100%
TRTV: Treatment group RTV; CRTV: negative control group RTV
Results: shown in table 4 and
The results of experiment of tumor growth inhibition induced by mPEG-SC20k-HM-3 on human gastric cancer MGC803 xenograft tumors in nude mice demonstrated that compared with negative control group, mPEG-SC20K-HM-3 36.7 mg/kg played a significant inhibition role in growth of human gastric cancer MGC803. *P<0.05 (there is significant difference compared with negative group).
Picked the tumor tissue of vigorous growth and was cut into pieces of about 1.5 mm3. Under sterile conditions, the tumor tissue was inoculated subcutaneously on the right of nude mice. Tumor diameter was measured with a Vernier caliper. The animals which had tumors with a size of 100-200 mm3 were randomly divided into groups. The effect of polypeptide on anti-tumor was dynamically observed through the method of measuring tumor diameter. The times of measuring tumor diameter were once 2 days, and meantime, weighed mice. The mice in experimental group were infused polypeptide through tail vein, once 3 days. The mice in the placebo group were injected saline with the same volume. Tumor volume formula:
TV=0.52×a×b2
a, b respectively represent the length and width. According to the result of measurement, calculate the relative tumor volume. Evaluation of antitumor activity is relative tumor proliferation rate T/C(%). The formula is as follows: T/C(%)=TRTV/CRTV×100%
TRTV:Treatment group RTV; CRTV: negative control group RTV
Results: shown in table 4 and
The results of experiment of tumor growth inhibition induced by mPEG-SC20k-HM-3 on human pancreatic cancer SW-1990 xenograft tumors in nude mice demonstrated that compared with negative control group, mPEG-SC20K-HM-3 36.7 mg/kg played a significant inhibition role in growth of human pancreatic cancer SW-1990. *P<0.05 (there is significant difference compared with negative group).
Picked the tumor tissue of vigorous growth and was cut into pieces of about 1.5 mm3. Under sterile conditions, the tumor tissue was inoculated subcutaneously on the right of nude mice. Tumor diameter was measured with a vernier caliper. The animals which had tumors with a size of 100-200 mm3 were randomly divided into groups. The effect of polypeptide on anti-tumor was dynamically observed through the method of measuring tumor diameter. The times of measuring tumor diameter were once 2 days, and meantime, weighed mice. The mice in experimental group were infused polypeptide through tail vein, once 3 days. The mice in the placebo group were injected saline with the same volume. Tumor volume formula:
TV=0.52×a×b2
a, b respectively represent the length and width. According to the result of measurement, calculate the relative tumor volume. Evaluation of antitumor activity is relative tumor proliferation rate T/C(%). The formula is as follows: T/C(%)=TRTV/CRTV×100%
TRTV: Treatment group RTV; CRTV: negative control group RTV
Results: shown in table 7 and
The results of experiment of tumor growth inhibition induced by mPEG-SC20k-HM-3 on human lung cancer H460 xenograft tumors in nude mice demonstrated that compared with negative control group, mPEG-SC20K-HM-3 36.7 mg/kg played a significant inhibition role in growth of human lung cancer H460. *P<0.05 (there is significant difference compared with negative group).
Picked the tumor tissue of vigorous growth and was cut into pieces of about 1.5 mm3. Under sterile conditions, the tumor tissue was inoculated subcutaneously on the right of nude mice. Tumor diameter was measured with a vernier caliper. The animals which had tumors with a size of 100-200 mm3 were randomly divided into groups. The effect of polypeptide on anti-tumor was dynamically observed through the method of measuring tumor diameter. The times of measuring tumor diameter were once 2 days, and meantime, weighed mice. The mice in experimental group were infused polypeptide through tail vein, once 3 days. The mice in the placebo group were injected saline with the same volume. Tumor volume formula: TV=0.52×a×b2
a, b respectively represent the length and width. According to the result of measurement, calculate the relative tumor volume. Evaluation of antitumor activity is relative tumor proliferation rate T/C(%). The formula is as follows: T/C(%)=TRTV/CRTV×100%
TRTV: Treatment group RTV; CRTV: negative control group RTV
Results: shown in table 8 and
The results of experiment of tumor growth inhibition induced by mPEG-SC20k-HM-3 on human breast cancer MDA-MB-231 xenograft tumors in nude mice demonstrated that compared with negative control group, mPEG-SC20K-HM-3 36.7 mg/kg played a significant inhibition role in growth of human breast cancer MDA-MB-231. *P<0.05 (there is significant difference compared with negative group).
Human gallbladder cancer GBC-SD cell lines in logarithmic growth phase were picked and made into 5×107/ml cell suspension of under sterile conditions, and then 0.1 ml suspension was inoculated subcutaneously at the right armpit of nude mice. Tumor diameter was measured with a vernier caliper. The animals which had tumors with a size of 100-200 mm3 were randomly divided into groups. The effect of polypeptide on anti-tumor was dynamically observed through the method of measuring tumor diameter. The times of measuring tumor diameter were once 2 days, and meantime, weighed mice. The mice in experimental group were infused polypeptide through tail vein. The mice in the placebo group were injected saline with the same volume, once 1 day; Taxol group: 10 mg/kg, once 1 week; Endostar group: 2.5 mg/kg, once 1 day; The groups of high, intermediate and low polypeptide: 6, 3, 1.5 mg/kg, respectively, once 1 day. After administration, all mice were sacrificed. Stripped the tumors and weighed.
Results: shown in table 9 and
The results of experiment of tumor growth inhibition induced by mPEG-SC20k-HM-3 on human gallbladder cancer GBC-SD xenograft tumors in nude mice demonstrated that compared with negative control group, mPEG-SC20K-HM-3 36.7 mg/kg played a significant inhibition role in growth of human gallbladder cancer GBC-SD. *P<0.05 (there is significant difference compared with negative group).
Human kidney cancer A498 cell lines in logarithmic growth phase were picked and made into 5×107/ml cell suspension of under sterile conditions, and then 0.1 ml suspension was inoculated subcutaneously at the right armpit of nude mice. Tumor diameter was measured with a vernier caliper. The animals which had tumors with a size of 100-200 mm3 were randomly divided into groups. The effect of polypeptide on anti-tumor was dynamically observed through the method of measuring tumor diameter. The times of measuring tumor diameter were once 2 days, and meantime, weighed mice. The mice in experimental group were infused polypeptide through tail vein. The mice in the placebo group were injected saline with the same volume, once 1 day; Taxol group: 10 mg/kg, once 1 week; Endostar group: 2.5 mg/kg, once 1 day; The groups of high, intermediate and low polypeptide: 6, 3, 1.5 mg/kg, respectively, once 1 day. After administration, all mice were sacrificed. Stripped the tumors and weighed.
Results: shown in table 10 and
The results of experiment of tumor growth inhibition induced by mPEG-SC20k-HM-3 on human kidney cancer A498 xenograft tumors in nude mice demonstrated that compared with negative control group, mPEG-SC20K-HM-3 36.7 mg/kg played a significant inhibition role in growth of human kidney cancer A498. *P<0.05 (there is significant difference compared with negative group).
Human colon cancer HT-29 cell lines in logarithmic growth phase were picked, and made into 5×107/ml cell suspension of under sterile conditions, and then 0.1 ml suspension was inoculated subcutaneously at the right armpit of nude mice. Tumor diameter was measured with a vernier caliper. The animals which had tumors with a size of 100-200 mm3 were randomly divided into groups. The effect of polypeptide on anti-tumor was dynamically observed through the method of measuring tumor diameter. The times of measuring tumor diameter were once 2 days, and meantime, weighed mice. The mice in experimental group were infused polypeptide through tail vein. The mice in the placebo group were injected saline with the same volume, once 1 day; Taxol group: 10 mg/kg, once 1 week; Endostar group: 2.5 mg/kg, once 1 day; The groups of high, intermediate and low polypeptide: 6, 3, 1.5 mg/kg, respectively, once 1 day. After administration, all mice were sacrificed. Stripped the tumors and weighed.
Results: shown in table 11 and
The results of experiment of tumor growth inhibition induced by mPEG-SC20k-HM-3 on human colon cancer HT-29 xenograft tumors in nude mice demonstrated that compared with negative control group, mPEG-SC20K-HM-3 36.7 mg/kg played a significant inhibition role in growth of human colon cancer HT-29. *P<0.05 (there is significant difference compared with negative group).
Human ovarian cancer SK-OV-3 cell lines in logarithmic growth phase were picked, and made into 5×107/ml cell suspension of under sterile conditions, and then 0.1 ml suspension was inoculated subcutaneously at the right armpit of nude mice. Tumor diameter was measured with a vernier caliper. The animals which had tumors with a size of 100-200 mm3 were randomly divided into groups. The effect of polypeptide on anti-tumor was dynamically observed through the method of measuring tumor diameter. The times of measuring tumor diameter were once 2 days, and meantime, weighed mice. The mice in experimental group were infused polypeptide through tail vein. The mice in the placebo group were injected saline with the same volume, once 1 day; Cisplatin group: 10 mg/kg, once 1 week; Endostar group: 2.5 mg/kg, once 1 day; The groups of high, intermediate and low polypeptide: 6, 3, 1.5 mg/kg, respectively, once 1 day. After administration, all mice were sacrificed. Stripped the tumors and weighed.
Results: shown in table 12 and
Therefore, the results of experiment of tumor growth inhibition induced by mPEG-SC20k-HM-3 on human ovarian cancer SK-OV-3 xenograft tumors in nude mice demonstrated that compared with negative control group, mPEG-SC20K-HM-3 36.7 mg/kg played a significant inhibition role in growth of human ovarian cancer SK-OV-3. *P<0.05 (there is significant difference compared with negative group).
Human endometrial cancer HHUA cell lines in logarithmic growth phase were picked, and made into 5×107/ml cell suspension of under sterile conditions, and then 0.1 ml suspension was inoculated subcutaneously at the right armpit of nude mice. Tumor diameter was measured with a vernier caliper. The animals which had tumors with a size of 100-200 mm3 were randomly divided into groups. The effect of polypeptide on anti-tumor was dynamically observed through the method of measuring tumor diameter. The times of measuring tumor diameter were once 2 days, and meantime, weighed mice. The mice in experimental group were infused polypeptide through tail vein. The mice in the placebo group were injected saline with the same volume, once 1 day; Taxol group: 10 mg/kg, once 1 week; Endostar group: 2.5 mg/kg, once 1 day; The groups of high, intermediate and low polypeptide: 6, 3, 1.5 mg/kg, respectively, once 1 day. After administration, all mice were sacrificed. Stripped the tumors and weighed.
Results: shown in table 13 and
Therefore, the results of experiment of tumor growth inhibition induced by mPEG-SC20k-HM-3 on human endometrial cancer HHUA xenograft tumors in nude mice demonstrated that compared with negative control group, mPEG-SC20K-HM-3 36.7 mg/kg played a significant inhibition role in growth of human endometrial cancer HHUA. *P<0.05 (there is significant difference compared with negative group).
Human cervical cancer HeLa cell lines in logarithmic growth phase were picked, and made into 5×107/ml cell suspension of under sterile conditions, and then 0.1 ml suspension was inoculated subcutaneously at the right armpit of nude mice. Tumor diameter was measured with a vernier caliper. The animals which had tumors with a size of 100-200 mm3 were randomly divided into groups. The effect of polypeptide on anti-tumor was dynamically observed through the method of measuring tumor diameter. The times of measuring tumor diameter were once 2 days, and meantime, weighed mice. The mice in experimental group were infused polypeptide through tail vein. The mice in the placebo group were injected saline with the same volume, once 1 day; Taxol group: 10 mg/kg, once 1 week; Endostar group: 2.5 mg/kg, once 1 day; The groups of high, intermediate and low polypeptide: 6, 3, 1.5 mg/kg, respectively, once 1 day. After administration, all mice were sacrificed. Stripped the tumors and weighed.
Results: shown in table 14 and
Therefore, the results of experiment of tumor growth inhibition induced by mPEG-SC20k-HM-3 on human cervical cancer HeLa xenograft tumors in nude mice demonstrated that compared with negative control group, mPEG-SC20K-HM-3 36.7 mg/kg played a significant inhibition role in growth of human cervical cancer HeLa. *P<0.05 (there is significant difference compared with negative group)
Human prostate cancer DU-145 cell lines in logarithmic growth phase were picked, and made into 5×107/ml cell suspension of under sterile conditions, and then 0.1 ml suspension was inoculated subcutaneously at the right armpit of nude mice. Tumor diameter was measured with a vernier caliper. The animals which had tumors with a size of 100-200 mm3 were randomly divided into groups. The effect of polypeptide on anti-tumor was dynamically observed through the method of measuring tumor diameter. The times of measuring tumor diameter were once 2 days, and meantime, weighed mice. The mice in experimental group were infused polypeptide through tail vein. The mice in the placebo group were injected saline with the same volume, once 1 day; Cisplatin group: 10 mg/kg, once 1 week; Endostar group: 2.5 mg/kg, once 1 day; The groups of high, intermediate and low polypeptide: 6, 3, 1.5 mg/kg, respectively, once 1 day. After administration, all mice were sacrificed. Stripped the tumors and weighed.
Results: shown in table 15 and
Therefore, the results of experiment of tumor growth inhibition induced by mPEG-SC20k-HM-3 on human prostate cancer DU-145 xenograft tumors in nude mice demonstrated that compared with negative control group, mPEG-SC20K-HM-3 36.7 mg/kg played a significant inhibition role in growth of human prostate cancer DU-145. *P<0.05 (there is significant difference compared with negative group).
Human bladder cancer HT1376 cell lines in logarithmic growth phase were picked, and made into 5×107/ml cell suspension of under sterile conditions, and then 0.1 ml suspension was inoculated subcutaneously at the right armpit of nude mice. Tumor diameter was measured with a vernier caliper. The animals which had tumors with a size of 100-200 mm3 were randomly divided into groups. The effect of polypeptide on anti-tumor was dynamically observed through the method of measuring tumor diameter. The times of measuring tumor diameter were once 2 days, and meantime, weighed mice. The mice in experimental group were infused polypeptide through tail vein. The mice in the placebo group were injected saline with the same volume, once 1 day; Taxol group: 10 mg/kg, once 1 week; Endostar group: 2.5 mg/kg, once 1 day; The groups of high, intermediate and low polypeptide: 6, 3, 1.5 mg/kg, respectively, once 1 day. After administration, all mice were sacrificed. Stripped the tumors and weighed.
Results: shown in table 16 and
Therefore, the results of experiment of tumor growth inhibition induced by mPEG-SC20k-HM-3 on human bladder cancer HT1376 xenograft tumors in nude mice demonstrated that compared with negative control group, mPEG-SC20K-HM-3 36.7 mg/kg played a significant inhibition role in growth of human bladder cancer HT1376. *P<0.05 (there is significant difference compared with negative group).
Human testicular cancer 5637 cell lines in logarithmic growth phase were picked, and made into 5×107/ml cell suspension of under sterile conditions, and then 0.1 ml suspension was inoculated subcutaneously at the right armpit of nude mice. Tumor diameter was measured with a vernier caliper. The animals which had tumors with a size of 100-200 mm3 were randomly divided into groups. The effect of polypeptide on anti-tumor was dynamically observed through the method of measuring tumor diameter. The times of measuring tumor diameter were once 2 days, and meantime, weighed mice. The mice in experimental group were infused polypeptide through tail vein. The mice in the placebo group were injected saline with the same volume, once 1 day; Cisplatin group: 10 mg/kg, once 1 week; Endostar group: 2.5 mg/kg, once 1 day; The groups of high, intermediate and low polypeptide: 6, 3, 1.5 mg/kg, respectively, once 1 day. After administration, all mice were sacrificed. Stripped the tumors and weighed.
Results: shown in table 17 and
Therefore, the results of experiment of tumor growth inhibition induced by mPEG-SC20k-HM-3 on human testicular cancer 5637 xenograft tumors in nude mice demonstrated that compared with negative control group, mPEG-SC20K-HM-3 36.7 mg/kg played a significant inhibition role in growth of human testicular cancer 5637. *P<0.05 (there is significant difference compared with negative group).
Sarcoma HT-1080 cell lines in logarithmic growth phase were picked, and made into 5×107/ml cell suspension of under sterile conditions, and then 0.1 ml suspension was inoculated subcutaneously at the right armpit of nude mice. Tumor diameter was measured with a vernier caliper. The animals which had tumors with a size of 100-200 mm3 were randomly divided into groups. The effect of polypeptide on anti-tumor was dynamically observed through the method of measuring tumor diameter. The times of measuring tumor diameter were once 2 days, and meantime, weighed mice. The mice in experimental group were infused polypeptide through tail vein. The mice in the placebo group were injected saline with the same volume, once 1 day; Cyclophosphamide group: 15 mg/kg, once 1 week; Polypeptide group: 3 mg/kg, once 1 day. After administration, all mice were sacrificed. Stripped the tumors and weighed.
Results: shown in table 18 and
Therefore, the results of experiment of tumor growth inhibition induced by mPEG-SC20k-HM-3 on sarcoma HT-1080 xenograft tumors in nude mice demonstrated that compared with negative control group, mPEG-SC20K-HM-3 36.7 mg/kg played a significant inhibition role in growth of sarcoma HT-1080. *P<0.05 (there is significant difference compared with negative group).
This application is a Divisional Application of U.S. patent application No. 14/359,462, filed May 20, 2014 entitled POLYETHYLENE GLYCOL-MODIFIED INTEGRIN BLOCKER HM-3 AND USE THEREOF which claims priority to PCT Application No. PCT/CN2012/084788, having a filing date of Nov. 17, 2012, based off of CN Application No. 201110370529.9, having a filing date of Nov. 21, 2011, the entire contents of which are hereby incorporated by reference.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 14359462 | May 2014 | US |
| Child | 15332539 | US |
