Polyethylene glycol-cactus oligopeptide bonding rapamycin derivatives
10098870 ยท 2018-10-16
Assignee
Inventors
- Jianhuan Jia (Beijing, CN)
- Zewang Feng (Beijing, CN)
- Jinliang Wang (Beijing, CN)
- Yan Liu (Beijing, CN)
- Zhenguo Wang (Beijing, CN)
- Xuan Zhao (Beijing, CN)
Cpc classification
A61K31/436
HUMAN NECESSITIES
A61P29/00
HUMAN NECESSITIES
A61K47/65
HUMAN NECESSITIES
A61P9/14
HUMAN NECESSITIES
A61P37/06
HUMAN NECESSITIES
A61K47/60
HUMAN NECESSITIES
International classification
A61K31/436
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
Abstract
The present invention provides compounds represented by formula (I) and pharmaceutical acceptable salts thereof, preparation method therefor and pharmaceutical composition containing the compounds represented by formula (I) and pharmaceutical acceptable salts thereof. In the compounds of the present invention, each terminal group of polyethylene glycol molecule can bond with a plurality of rapamycin molecules by cactus oligopeptide, with the loading rate of the pharmaceutical being increased. The compounds can be used to induce immunosuppression and treat graft rejection, autoimmune disease, solid tumors, fungal infection, and cardiovascular and cerebrovascular disease. ##STR00001##
Claims
1. A compound or a pharmaceutically acceptable salt thereof, wherein the compound comprises rapamycin conjugated to polyethylene glycol (PEG) and has the following formula: ##STR00027## and wherein the PEG has a number average molecular weight of 20,000 Daltons.
2. A method of treating and/or inhibiting graft rejection in a subject in need thereof, the method comprising administering to the subject the compound of claim 1 or a pharmaceutically acceptable salt thereof.
3. A method of treating rheumatoid arthritis in a subject in need thereof, the method comprising administering to the subject the compound of claim 1 or a pharmaceutically acceptable salt thereof.
4. A method of treating solid tumor in a subject in need thereof, the method comprising administering to the subject the compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein the solid tumor is selected from the group consisting of astrocytoma, liver cancer, prostate cancer, breast cancer, lung cancer and ovarian cancer.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION OF THE INVENTION
(13) Rapamycin shows good prospects in clinical application, but it still has a low bioavailability (<15%), poor water-solubility and other defects, a number of rapamycin derivatives with a high efficiency and specificity such as everolimus, temsirolimus, ridaforolimus and the like were further developed in the late 1990s, which are obtained by introducing polar groups into molecule of rapamycin to achieve the goal of enhancing the water-solubility of rapamycin. Rapamycin is so sensitive to acids and bases that it will be degraded even under physiological conditions, and the products obtained after degradation have no immunosuppressive activity, and it is a problem to be solved that how to increase the stability.
(14) Rapamycin, due to the hydroxyl groups in its structure, can be esterified with polyethylene glycol-cactus oligopeptide to form a prodrug to obtain an improved water-solubility, accelerated distribution of drug molecules, as well as a better permeability to tumor to avoid an allergic reaction induced by local aggregation of the drug; additionally, the polyethylene glycol fragment can form a hydrophilic barrier to prevent an excessive degradation of rapamycin, thus to obtain a rational use of rapamycin. Furthermore, a molecular conformation which is similar to but better than liposome is formed by chemically bonding of the drug with the amphiphilic substance to obtain an increase bioavailability of drug, reduced dosage, reduced side effects and prolonged duration of action by using the targeting towards tumors. Particularly, the pharmaceutically active ingredient is released by biodegradation of the ester group in vivo. The drug improved by this method has a good water-solubility, rapid onset, long duration and effective therapeutic effect.
(15) Unlike the PEGylated rapamycin derivative in patent WO2007/103348, a cactus oligopeptide is used to enable rapamycin to bond with polyethylene glycol. The oligopeptide used in the present invention refers to a polypeptide comprising 2-12 amino acids, which can be completely hydrolyzed to be free amino acids by peptidase and enter the bloodstream in the form of free amino acids. Amino acids have a good biocompatibility, and is dispersible in vivo and safe after biodegradation, at the same time, the cactus oligopeptide can provide more reactive sites, a larger loading rate to bond with more drug moleculars and to increase the range of choice of polyethylene glycol used.
(16) The conjugates according to the present invention may be administered in the form of pure compound or suitable pharmaceutical compositions with any acceptable modes of administration or regents for similar application. Thus, the conjugates according to the present invention may be administered orally, nasally, parenterally, topically, transdermally or rectally, in the form of solid, semi-solid, lyophilized powder, or liquid medicaments, e.g., tablets, capsules, pills, granules, powders, suppositories, injections, solutions, suspensions, ointments, patches, lotions, drops, liniments, aerosols, etc. The unit dosage forms which are suitable for precise and simple administration are preferred. The composition may contain conventional pharmaceutical carriers or excipients and conjugates according to the present invention as active ingredients (one or more), as well as other medicaments, carriers and adjuvants etc.
(17) Generally, according to the desired mode of administration, the pharmaceutically acceptable composition contains the conjugate according to the present invention with a weight percentage of about 1 to about 99 and a suitable pharmaceutical excipient with a weight percentage of about 99 to 1. The composition comprising conjugate according to the present invention with a weight percentage of about 5 to 75 with the rest being a suitable pharmaceutical excipient is preferred.
(18) The pharmaceutical compositions may be administered in liquid form, e.g. by dissolving or dispersing the conjugates according to the present invention (from about 0.5 to about 20%) and pharmaceutically acceptable adjuvants which are employed selectively into carriers to thereby form a solution or suspension, the examples of carrier are water, saline, glucose hydrate, glycerol and ethanol etc.
(19) If necessary, the pharmaceutical compositions according to the present invention may also contain minor amounts of auxiliary substances such as wetting agents or emulsifiers, pH buffers, antioxidants, etc., for example: citric acid, sorbitan monolaurate, triethanolamine oleate and butylated hydroxy toluene, etc.
(20) The following examples are used to illustrate the present invention but are not used to limit the present invention.
EXAMPLE
(21) Rapamycin and L-(+)-glutamic acid used in the embodiments are purchased from Wuhan Yuanchenggongchuang Technology Co., Ltd. and Beijing Chemical Reagent Company, respectively, tert-butyl bromoacetate, triphenylphosphine, p-toluenesulfonic acid, benzyl alcohol and dicyclohexylcarbodiimide (DCC) are purchased from Sinopharm Chemical Reagent Co., Ltd., 4-dimethylaminopyridine (DMAP) and 1-hydroxy benzotriazole (HOBt) are purchased from Shanghai MEDPEP Co., Ltd., N-t-butoxycarbonyl-L-glutamic acid-5-benzyl ester is purchased from Sichuan Tongsheng Amino Acid Co., Ltd., monomethoxy polyethylene glycol acetic acid, monomethoxy polyethylene glycol-glutamic acid dipeptide, Y-type polyethylene glycol acetic acid are provided by Beijing Jenkem Technology Co., Ltd., other reagents are commercially available.
Example 1 Preparation of Glycine Ester of Rapamycin
(22) ##STR00021## ##STR00022##
(23) Tert-butyl bromoacetate (5.82 g, 30 mmoL) was added to the reaction flask and dissolved by acetone (80 mL), a solution obtained by sodium azide (4.55 g, 70 mmoL) dissolved in water (40 mL) was then added, the mixture obtained was heated and refluxed overnight. Acetone was distilled off the reaction solution, the residue was extracted with ether, the extract obtained was washed by saturated brine, dried and concentrated under reduced pressure to give an oily liquid. This liquid was dissolved by methanol (90 mL) and added by 1 N of sodium hydroxide solution (90 mL), stirred, heated and refluxed for 3 h. After being cooled, methanol was distilled off under reduced pressure, the residue was cooled by an ice bath and added by 6 N of hydrochloric acid to adjust the value of pH to 2, and then extracted with ether, the extract obtained was washed by water, dried, and concentrated to give azidoacetic acid, MS m/z: 124 [M+Na].sup.+.
(24) Azidoacetic acid (253 mg, 2.5 mmoL) and rapamycin (2.28 g, 2.5 mmoL) were added to the reaction flask, dissolved with dichloromethane, cooled by an ice bath, and then 4-dimethylaminopyridine (DMAP, 611 mg, 5 mmoL) and N,N-dicyclohexylcarbodiimide (DCC, 1.03 g, 5 mmoL) were added to the reaction flask, the mixture was continued to be stirred at room temperature overnight after the addition. The residue obtained after concentration of the reaction solution was purified by column chromatography to give 1.42 g of azide acetate of rapamycin with a yield of 57%, MS m/z: 1020 [M+Na]+.
(25) Azide acetate of rapamycin (0.7 g, 0.7 mmoL) and triphenylphosphine (0.37 g, 1.4 mmoL) were added to the reaction flask, then a mixture of tetrahydrofuran and water (5:1, 180 mL) was added, the reaction was heated to 50 C. overnight, the residue obtained after the concentration of reaction solution was extracted with ethyl acetate, the extract obtained was washed by saturated brine, dried. The residue obtained after concentration under reduced pressure was purified by column chromatography to give 0.48 g of glycine ester of rapamycin with a yield of 70%, MS m/z: 994 [M+Na]+.
Example 2 Preparation of Monomethoxy Polyethylene Glycol (with a Number Average Molecular Weight of 20,000)-Rapamycin Conjugate (LPR-1)
(26) ##STR00023##
(27) Monomethoxy polyethylene glycol acetic acid (20 K, 1 g, 0.05 mmoL), glycine ester of rapamycin (97 mg, 0.1 mmoL) prepared in Example 1, 1-hydroxy benzotriazole (HOBt, 6.8 mg, 0.05 mmoL) and DMAP (12.2 mg, 0.1 mmoL) were added to the reaction flask, dissolved with dichloromethane, cooled by an ice bath, then added dropwise by a solution obtained by DCC (15.5 mg, 0.075 mmoL) dissolved in dichloromethane, warmed to room temperature naturally after the dropping, the reaction was kept overnight, the next day the reaction solution was concentrated and the residue was crystallized with isopropanol to give 0.82 g of monomethoxy polyethylene glycol (20 K)-rapamycin conjugate (LPR-1) (n is about 450).
(28) .sup.1H-NMR (300 MHz, CDCl.sub.3): 0.90 (Me, 3H, 43), 0.92 (Me, 3H, 49), 0.94 (Me, 3H, 46), 0.96 (Me, 3H, 48), 0.97 (Me, 3H, 45), 1.10 (CH.sub.2, 2H, 24), 1.11 (CH.sub.2, 2H, 36), 1.20 (CH.sub.2, 2H, 42), 1.33 (CH.sub.2, 2H, 41), 1.37 (CH, 1H, 37), 1.45 (CH.sub.2, 2H, 5), 1.47 (CH.sub.2, 2H, 4), 1.60 (CH.sub.2, 2H, 13), 1.61 (CH.sub.2, 2H, 12), 1.65 (CH.sub.2, 2H, 15), 1.65 (CH.sub.2, 2H, 44), 1.74 (Me, 3H, 47), 1.75 (CH, 1H, 35), 2.07 (CH, 4H, 3, 11, 23, 25), 2.08 (CH.sub.2, 2H, 33), 3.14 (Me, 3H, 50), 3.33 (CH, 1H, 31), 3.36 (Me, 3H, 51), 3.37 (CH.sub.2, 2H, 6), 3.42 (CH, 1H, 40), 3.44 (Me, 3H, 52), 3.56 (CH, 1H, 39), 3.64 (CH.sub.2, 1800H, PEG), 3.71 (CH, 1H, 16), 3.72 (CH, 1H, 27), 3.86 (CH, 1H, 14), 4.17 (CH.sub.2, 2H, 54), 4.19 (CH, 1H, 28), 5.16 (CH, 1H, 2), 5.17 (CH, 1H, 34), 5.29 (CH, 1H, 30), 5.39 (CH, 1H, 22), 5.95 (CH, 1H, 18), 6.13 (CH, 1H, 21), 6.31 (CH, 1H, 20), 6.38 (CH, 1H, 19), 8.34 (CH, 1H, 55).
Example 3 Preparation of Monomethoxy Polyethylene Glycol (with a Number Average Molecular Weight of 20,000)-Glutamic Acid Dipeptide-Rapamycin Conjugate (LPR-2)
(29) ##STR00024##
(30) Monomethoxy polyethylene glycol-glutamic acid dipeptide (20 K, 0.5 g, 0.025 mmol), glycine ester of rapamycin 48.6 mg (0.05 mmoL) prepared in Example 1, HOBt (3.4 mg, 0.025 mmoL) and DMAP 6.1 mg (0.05 mmoL) were added to the reaction flask, dissolved with dichloromethane, cooled by an ice bath, then added dropwise by a solution obtained by DCC 15.5 mg (0.075 mmoL) dissolved in dichloromethane, warmed to room temperature naturally after the dropping, the reaction was kept overnight. The next day the reaction solution was concentrated and the residue was crystallized with isopropanol to give 0.41 g of monomethoxy polyethylene glycol (20K)-glutamic acid dipeptide-rapamycin conjugate (LPR-2) (n is about 450).
(31) .sup.1H-NMR (300 MHz, CDCl.sub.3): 0.90 (Me, 9H, 43), 0.92 (Me, 9H, 49), 0.94 (Me, 9H, 46), 0.96 (Me, 9H, 48), 0.97 (Me, 9H, 45), 1.10 (CH.sub.2, 6H, 24), 1.11 (CH.sub.2, 6H, 36), 1.20 (CH.sub.2, 6H, 42), 1.33 (CH.sub.2, 6H, 41), 1.37 (CH, 3H, 37), 1.45 (CH.sub.2, 6H, 5), 1.47 (CH.sub.2, 6H, 4), 1.60 (CH.sub.2, 6H, 13), 1.61 (CH.sub.2, 6H, 12), 1.65 (CH.sub.2, 6H, 15), 1.65 (CH.sub.2, 6H, 44), 1.74 (Me, 9H, 47), 1.75 (CH, 3H, 35), 2.07 (CH, 12H, 3, 11, 23, 25), 2.08 (CH.sub.2, 6H, 33), 3.14 (Me, 9H, 50), 3.33 (CH, 3H, 31), 3.36 (Me, 9H, 51), 3.37 (CH.sub.2, 6H, 6), 3.42 (CH, 3H, 40), 3.44 (Me, 9H, 52), 3.56 (CH, 3H, 39), 3.64 (CH.sub.2, 1800H, PEG), 3.71 (CH, 3H, 16), 3.72 (CH, 3H, 27), 3.86 (CH, 3H, 14), 4.17 (CH.sub.2, 6H, 54), 4.19 (CH, 3H, 28), 5.16 (CH, 3H, 2), 5.17 (CH, 3H, 34), 5.29 (CH, 3H, 30), 5.39 (CH, 3H, 22), 5.95 (CH, 3H, 18), 6.13 (CH, 3H, 21), 6.31 (CH, 3H, 20), 6.38 (CH, 3H, 19), 8.34 (CH, 3H, 55).
Example 4 Preparation of Y-Type Polyethylene Glycol (with a Number Average Molecular Weight of 40,000)-Glutamic Acid Dipeptide-Rapamycin Conjugate (LPR-3)
(32) ##STR00025## ##STR00026##
(33) N-t-butoxycarbonyl-benzyl glutamate dipeptide (0.78 g, 1.2 mmoL) (Example 3) was dissolved in dichloromethane (10 mL), added by 3 mL of trifluoroacetic acid, the reaction was kept at room temperature for 2 h. 100 mL of dichloromethane was added after removal of solvent, and sodium bicarbonate solution with a concentration of 5% was added to adjust the value of pH to 7-8. The reaction mixture was extracted and separated, the organic phase was washed with sodium bicarbonate solution with a concentration of 5% twice, dried with anhydrous sodium sulfate. The filtrate obtained after filtration was added directly to the reaction flask, and Y-type polyethylene glycol acetic acid (40 K, 40.0 g, 1 mmoL), DMAP (245 mg, 2 mmol), HOBt (135 mg, 1 mmol) were added under the protection of nitrogen. After reactants being completely dissolved, DCC (412 mg, 2 mmol) was added. The reaction was stirred at room temperature overnight. The reaction mixture was filtered and rotary evaporated to remove the solvent, the residue obtained was added by 500 mL of isopropanol, filtered, and the product obtained was dried under vacuum. This product was dissolved in 200 mL of anhydrous methanol, added by 1.0 g of palladium on carbon and introduced by hydrogen overnight at room temperature. Palladium on carbon was removed by filtration, the solvent was removed by rotary evaporation, the residue obtained was added into 500 mL of isopropanol, filtered and dried under vacuum. 33.4 g of Y-type polyethylene glycol-glutamic acid dipeptide (40 K) was obtained.
(34) Y-type polyethylene glycol-glutamic acid dipeptide (40 K, 0.5 g, 0.0125 mmol), glycine ester of rapamycin 24.3 mg (0.025 mmol) prepared in Example 1, HOBt (1.7 mg, 0.0125 mmo) and DMAP 3 mg (0.025 mmol) were added to the reaction flask, dissolved with dichloromethane, cooled by an ice bath, then added dropwise by a solution obtained by DCC 4.1 mg (0.02 mmol) dissolved in dichloromethane, the mixture was warmed to room temperature naturally after the dropping, the reaction was kept overnight. The next day the reaction solution was concentrated and the residue was crystallized with isopropanol to give 0.44 g of Y-type polyethylene glycol (40K)-glutamic acid dipeptide-rapamycin conjugate (LPR-3) (n is about 450).
(35) 1H-NMR (300 MHz, CDCl3): 0.90 (Me, 9H, 43), 0.92 (Me, 9H, 49), 0.94 (Me, 9H, 46), 0.96 (Me, 9H, 48), 0.97 (Me, 9H, 45), 1.10 (CH2, 6H, 24), 1.11 (CH2, 6H, 36), 1.20 (CH2, 6H, 42), 1.33 (CH2, 6H, 41), 1.37 (CH, 3H, 37), 1.45 (CH2, 6H, 5), 1.47 (CH2, 6H, 4), 1.60 (CH2, 6H, 13), 1.61 (CH2, 6H, 12), 1.65 (CH2, 6H, 15), 1.65 (CH2, 6H, 44), 1.74 (Me, 9H, 47), 1.75 (CH, 3H, 35), 2.07 (CH, 12H, 3, 11, 23, 25), 2.08 (CH2, 6H, 33), 3.14 (Me, 9H, 50), 3.33 (CH, 3H, 31), 3.36 (Me, 9H, 51), 3.37 (CH2, 6H, 6), 3.42 (CH, 3H, 40), 3.44 (Me, 9H, 52), 3.56 (CH, 3H, 39), 3.64 (CH2, 1800H, PEG), 3.71 (CH, 3H, 16), 3.72 (CH, 3H, 27), 3.86 (CH, 3H, 14), 4.17 (CH2, 6H, 54), 4.19 (CH, 3H, 28), 5.16 (CH, 3H, 2), 5.17 (CH, 3H, 34), 5.29 (CH, 3H, 30), 5.39 (CH, 3H, 22), 5.95 (CH, 3H, 18), 6.13 (CH, 3H, 21), 6.31 (CH, 3H, 20), 6.38 (CH, 3H, 19), 8.34 (CH, 3H, 55).
Example 5 the Inhibitory Activity of Different Polyethylene Glycol-Cactus Oligopeptide-Rapamycin Conjugates Against Tumor Cells
(36) (1) Experimental Method and Procedure
(37) (a) Cell Culture
(38) Plc/prf/5 cells were cultured with a monolayer in vitro in MEM medium supplied with heat-inactivated fetal bovine serum with a volume ratio of 10%, and an incubator at 37 C. with the air containing CO.sub.2 with a proportion of 5%. The tumor cells were passaged with digestion by trypsin-EDTA twice a week. The cells in the exponential growth phase were collected, counted, and used for inoculation.
(39) (b) Inoculation of Tumor Cells, Grouping and Administration
(40) 110.sup.7 of plc/prf/5 tumor cells were suspended in 0.1 ml of mixed solution (PBS:Matrigel=4:1), inoculated to each NOD/SCID mouse at the right shoulder. 24 days later the mean tumor volume was desired to reach about 350 mm.sup.3, the mice with a smaller or larger tumor were removed and the remaining mice were divided into groups randomly according to tumor size and administrated.
(41) (c) Experimental Scheme
(42) TABLE-US-00001 TABLE 1 The grouping and dosage regimen of experimental animals Compound used Dosage .sup.a Dosing volume Route of Dosage Group N for treatment (mg/kg) (l/g) administration regimen 1 5 Vehicle control 10 i.v. Q2W 2W 2 5 LPR-1 10 mg/kg 10 i.v. Q2W 2W 3 5 LPR-2 10 mg/kg 10 i.v. Q2W 2W 4 5 LPR-3 10 mg/kg 10 i.v. Q2W 2W .sup.a The dosage is counted with rapamycin, the same below.
Wherein Q2W2W represents intravenous injection twice a week for 2 weeks, the same below.
(2) Experimental Results
(43) (a) Body Weight
(44) Changes in body weight of tumor-bearing mice in each treatment group are shown in Table 2 and
(45) TABLE-US-00002 TABLE 2 The body weight of each treatment group at different time points Body weight of animal (g).sup.a Days after LPR-1 LPR-2 LPR-3 inoculation Vehicle control 10 mg/kg 10 mg/kg 10 mg/kg 24 17.6 0.5 19.2 0.7 19.6 1.5 19.2 0.7 27 18.3 0.3 19.5 0.8 18.6 1.2 18.0 0.4 31 17.4 0.1 18.4 0.6 18.0 1.2 17.7 0.4 34 18.0 0.2 19.1 0.8 18.5 1.2 18.2 0.7 38 18.1 0.2 18.8 0.5 18.1 1.2 17.8 0.7 Note: .sup.amean value standard error
(46) (b) Tumor Volume
(47) Changes in tumor volume of each treatment group are shown in Table 3 and
(48) TABLE-US-00003 TABLE 3 The tumor volume of each treatment group at different time points Tumor volume (mm.sup.3).sup.a Days after LPR-1 LPR-2 LPR-3 inoculation Vehicle control 10 mg/kg 10 mg/kg 10 mg/kg 24 355 64 352 49 353 54 358 62 27 763 102 598 80 404 89 455 85 31 1048 104 670 74 391 72 569 100 34 1439 130 738 74 456 77 754 151 38 1801 162 919 78 536 77 848 178 Note: .sup.amean value standard error
(49) (c) Evaluation of Anti-Tumor Effect
(50) The evaluation indexes of anti-tumor effect of LPR 1,2,3 on subcutaneous transplantation tumor model of plc/prf/5 are shown in Table 4.
(51) TABLE-US-00004 TABLE 4 Evaluation of anti-tumor effect of each treatment group Tumor Days for delaying Tumor volume control rate tumor growth Group (mm.sup.3).sup.a T/C (%) (to 1000 mm.sup.3) P value Vehicle 1801 162 control LPR-1 919 78 51 7 0.000 (10 mg/kg) LPR-2 536 77 30 >7 0.000 (10 mg/kg) LPR-3 848 178 47 >7 0.000 (10 mg/kg) Note: .sup.amean value standard error
(3) Summary and Discussion of Experimental Results
(52) In the experiment, the pesticide effects in vivo of LPR-1, LPR-2 and LPR-3 on subcutaneous transplantation tumor model of human hepatoma cell plc/prf/5 were evaluated. The tumor volume of each treatment group at different time points is shown in Table 2 and
(53) Effect of changes in body weight of tumor-bearing mice in each treatment group is shown in Table 1 and
(54) In summary, in this study, the test drugs LPR-1, LPR-2 and LPR-3 showed an anti-tumor effect on subcutaneous transplantation tumor model of human hepatoma cell plc/prf/5, wherein LPR-2 showed obviously the maximum anti-tumor effect, no obvious toxic reaction of each treatment group was observed during the experiment. The polyethylene glycol used in LPR-1 and LPR-2 had the same structure and number average molecular weight, but in structure of LPR-1 polyethylene glycol bonded with rapamycin only through glycine molecular causing that each terminal group of polyethylene glycol bonding with only one rapamycin molecular; while in structure of LPR-2 polyethylene glycol bonded with rapamycin through glutamic acid dipeptide and glycine causing that each terminal group of polyethylene glycol bonding with three rapamycin molecular. LPR-2 had a drug loading rate 3 times as much as that of LPR-1, and anti-tumor effect significantly higher than that of LPR-1.
Example 6 the Inhibitory Activity of Monomethoxy Polyethylene Glycol (with a Number Average Molecular Weight of 20,000)-Glutamic Acid Dipeptide-Rapamycin Conjugate (LPR-2) and Reference Substance Against PLC/PRF/5 Hepatoma Cells
(55) (1) Experimental Method and Procedure
(56) (a) Cell Culture
(57) Plc/prf/5 cells were cultured with a monolayer in vitro in MEM medium supplied with heat-inactivated fetal bovine serum with a volume ratio of 10%, and an incubator at 37 C. with the air containing CO.sub.2 with a proportion of 5%. The tumor cells were passaged with digestion by trypsin-EDTA twice a week. The cells in the exponential growth phase were collected, counted, and used for inoculation.
(58) (b) Inoculation of Tumor Cells, Grouping and Administration
(59) 8.3210.sup.6 of plc/prf/5 tumor cells were suspended in 0.1 ml of mixed solution (PBS:Matrigel=6:4), inoculated to each nude mouse at the right shoulder, and there were totally 36 mice inoculated. 10 days later the mean tumor volume was desired to reach about 161 mm.sup.3, the mice with a smaller or larger tumor were removed and the remaining 24 mice were divided into groups randomly according to tumor volume and administrated.
(60) (c) Experimental Scheme
(61) TABLE-US-00005 TABLE 5 The grouping and dosage regimen of experimental animals Compound used Dosage Dosing volume Route of Dosage Group N for treatment (mg/kg) (l/g) administration regimen 1 6 Physiological saline 10 i.v. QW 4W 2 6 5-FU 25 10 i.v. QD 5.sup. 3 6 LPR-2 30 10 i.v. BIW 4W 4 6 LPR-2 45 15 i.v. QW 4W
Wherein QW4W represents intravenous injection once a week for 4 weeks, BIW4W represents intravenous injection once every two weeks for 4 weeks, the same below.
(2) Experimental Results
(62) (a) Body Weight
(63) LPR-2 and 5-FU had an effect on body weight of mice bearing xenograft tumor model of plc/prf/5.
(64) Changes in body weight of tumor-bearing mice in each treatment group are shown in Table 6 and
(65) TABLE-US-00006 TABLE 6 The body weight of each treatment group at different time points Body weight of animal (g).sup.a Days after Physiological 5-FU LPR-2 LPR-2 inoculation saline 25 mg/kg 30 mg/kg 45 mg/kg 10 19.4 0.5 19.6 0.3 19.9 0.5 19.8 0.5 13 19.9 0.4 18.4 0.5 19.6 0.8 18.9 0.5 17 19.4 0.2 14.7 0.5 20.7 0.7 20.2 0.3 20 19.5 0.2 20.4 0.6 19.7 0.3 24 19.7 0.4 20.6 0.7 21.1 0.3 27 20.0 0.2 20.5 0.6 20.7 0.2 31 19.8 0.2 20.1 0.6 21.0 0.2 .sup.34.sup.b 20.0 0.3 19.5 0.6 19.7 0.5 38 20.0 0.4 19.8 0.7 20.7 0.3 Note: .sup.amean value standard error; .sup.blast administration.
(66) (b) Tumor Growth
(67) Changes in tumor volume of each treatment group are shown in Table 7 and
(68) TABLE-US-00007 TABLE 7 The tumor volume of each treatment group at different time points Tumor volume (mm.sup.3).sup.a Days after Physiological 5-FU LPR-2 LPR-2 inoculation saline 25 mg/kg 30 mg/kg 45 mg/kg 10 171 18 170 16 171 15 171 19 13 269 37 118 7 176 11 180 25 17 507 77 69 4 217 25 234 28 20 645 105 260 30 257 24 24 886 130 303 32 399 25 27 1,119 156 317 42 496 39 31 1,299 154 395 39 607 60 .sup.34.sup.b 1,455 157 437 44 640 93 38 1,973 211 553 63 948 144 Note: .sup.amean value standard error; .sup.blast administration.
(69) (c) Evaluation of Anti-Tumor Effect
(70) The evaluation indexes of anti-tumor effect of LPR-2 and 5-FU on xenograft tumor model of plc/prf/5 are shown in Table 8.
(71) TABLE-US-00008 TABLE 8 Evaluation of anti-tumor effect of each treatment group Tumor volume Tumor weight TGD (mm.sup.3).sup.b RTV.sup.b (mg).sup.b (to 1,000 T/C (%) P value Group N.sup.a 10.sup.th day 38.sup.th day (38.sup.th day) (38.sup.th day) mm.sup.3) RTV TW.sup.c RTV TW.sup.c Physiological 6 171 18 1,973 211 12.05 1.63 1877 195 0 100 100 1.000 1.000 saline LPR-2 6 171 15 553 63 3.48 0.68 517 67 >12 29 27 0.000 0.000 (30 mg/kg) LPR-2 6 171 19 .sup.948 144 5.61 0.67 845 87 >12 46 45 0.001 0.000 (45 mg/kg) Note: .sup.athe number of surviving animals in each group after completion of the treatment; .sup.bMean SEM; .sup.cTW (Tumor Weight).
(3) Summary and Discussion of Experimental Results
(72) In the experiment, the pesticide effect in vivo of LPR-2 on subcutaneous transplantation tumor model of human hepatoma cell plc/prf/5 in immune deficiency mice was evaluated. The tumor volume of each treatment group at different time points is shown in Table 7 and
(73) The 5-FU positive control group (25 mg/kg) showed an obvious anti-tumor effect, but was so toxic with this dosage that all the animals died. Two groups of LPR-2 with different dosages (30 and 45 mg/kg) had significant anti-tumor effect, with tumor volume of 553 and 948 mm.sup.3, respectively, at the end of experiment, and T/C values of 29% and 46% and p values of 0.000 and 0.001, compared with the physiological saline group. The analysis result of tumor weight was consistent with the tumor volume.
(74) Effect of changes in body weight of tumor-bearing mice in each group is shown in Table 6 and
(75) In summary, in this study, the test drug LPR-2 with dosages of 30 and 45 mg/kg showed significant anti-tumor effect on xenograft tumor model of human hepatoma cell plc/prf/5, and the animals treated with LPR-2 showed a good tolerance and no death of animals occurred in the treatment group. The positive drug 5-FU had a significant toxicity and the whole group of animals died.
Example 7 the Inhibitory Activity of Monomethoxy Polyethylene Glycol (with a Number Average Molecular Weight of 20,000)-Glutamic Acid Dipeptide-Rapamycin Conjugate (LPR-2) and Reference Substance Against Hep3B Hepatoma Cells
(76) (1) Experimental Method and Procedure
(77) (a) Cell Culture
(78) Hep3B cells were cultured with a monolayer in vitro in MEM medium supplied with heat-inactivated fetal bovine serum with a volume ratio of 10%, and an incubator at 37 C. with the air containing CO.sub.2 with a proportion of 5%. The tumor cells were passaged with digestion by trypsin-EDTA twice a week. The cells in the exponential growth phase were collected, counted, and used for inoculation.
(79) (b) Inoculation of Tumor Cells, Grouping and Administration
(80) 5.6710.sup.6 of Hep3B tumor cells were suspended in 0.1 ml of mixed solution (PBS:Matrigel=7:3), inoculated to each mouse at the right shoulder, and there were totally 37 mice inoculated. 16 days later the mean tumor volume was desired to reach about 453 mm.sup.3, the mice with a smaller or larger tumor were removed and the remaining 24 mice were divided into groups randomly according to tumor volume and administrated.
(81) (c) Experimental Scheme
(82) TABLE-US-00009 TABLE 9 The grouping and dosage regimen of experimental animals Compound used Dosage Dosing volume Route of Dosage Group N for treatment (mg/kg) (l/g) administration regimen 1 6 Physiological saline 10 i.v. QW 4W 2 6 5-FU 15 10 i.v. (QD 5) 2W 3 6 LPR-2 30 10 i.v. BIW 4W 4 6 LPR-2 45 15 i.v. QW 4W
(2) Experimental Results
(83) (a) Body Weight
(84) LPR-2 and 5-FU had an effect on body weight of mice bearing xenograft tumor model of Hep3B.
(85) Changes in body weight of tumor-bearing mice in each treatment group are shown in Table 10 and
(86) TABLE-US-00010 TABLE 10 The weight of each treatment group at different time points Body weight of animal (g).sup.a LPR-2 LPR-2 Days after Physiological 5-FU 30 mg/kg 45 mg/kg inoculation saline 15 mg/kg BIW 4W QW 4W 16 20.1 0.4 20.7 0.4 20.2 0.6 19.2 0.5 20 19.7 0.4 19.4 0.4 19.7 0.4 18.6 0.4 23 19.6 0.5 19.7 0.3 20.4 0.5 18.6 0.4 27 19.2 0.6 18.6 0.3 19.7 0.5 17.9 0.4 30 18.6 0.4 18.1 0.3 19.1 0.5 18.2 0.4 34 18.2 0.4 18.5 0.4 19.1 0.6 18.2 0.4 37 18.0 0.5 18.3 0.3 18.6 0.4 18.0 0.3 .sup.41.sup.b 17.7 0.5 18.1 0.3 17.9 0.3 16.7 0.5 44 17.3 0.4 18.4 0.3 17.3 0.3 17.1 0.4 Note: .sup.amean value standard error; .sup.blast administration.
(87) (b) Tumor Growth
(88) Changes in tumor volume of each treatment group are shown in Table 11 and
(89) TABLE-US-00011 TABLE 11 The tumor volume of each treatment group at different time points Tumor volume (mm.sup.3).sup.a LPR-2 LPR-2 Days after Physiological 5-FU 30 mg/kg 45 mg/kg inoculation saline 15 mg/kg BIW 4W QW 4W 16 453 72 453 66 454 60 453 56 20 701 116 567 84 524 47 490 73 23 860 147 667 105 622 37 549 83 27 1,056 157 820 149 643 45 589 91 30 1,192 163 850 150 689 57 584 87 34 1,374 225 1,123 226 761 82 608 89 37 1,576 239 1,500 295 899 97 699 102 .sup.41.sup.b 1,775 274 1,793 342 995 126 733 93 44 1,984 317 2,114 395 1,074 130 827 112 Note: .sup.amean value standard error; .sup.blast administration.
(90) (c) Evaluation of Anti-Tumor Effect
(91) The evaluation indexes of anti-tumor effect of LPR-2 and 5-FU on xenograft tumor model of Hep3B are shown in Table 12.
(92) TABLE-US-00012 TABLE 12 Evaluation of anti-tumor effect of each treatment group Tumor volume Tumor weight TGD (mm.sup.3).sup.b RTV.sup.b (mg).sup.b (to 1,000 T/C (%) P value Group N.sup.a 16.sup.th day 44.sup.th day (44.sup.th day) (44.sup.th day) mm.sup.3) RTV TW.sup.c RTV TW.sup.c Physiological 6 453 72 1,984 317 5.00 0.95 1555 267 0 100 100 1.000 1.000 saline 5-Fu 6 453 66 2,114 395 4.53 0.32 1695 289 6 106 109 0.996 0.999 (15 mg/kg) LPR-2 6 454 60 1,074 130 2.43 0.19 720 94 17 54 46 0.177 0.113 (30 mg/kg) BIW 4W LPR-2 6 453 56 .sup.827 112 1.85 0.17 625 84 >17 42 40 0.089 0.075 (45 mg/kg) QW 4W Note: .sup.athe number of surviving animals in each group after completion of the treatment; .sup.bMean SEM; .sup.cTW (Tumor Weight).
(3) Summary and Discussion of Experimental Results
(93) In the experiment, the pesticide effect in vivo of LPR-2 on subcutaneous transplantation tumor model of human hepatoma cell Hep3B in immune deficiency mice was evaluated. The tumor volume of each treatment group at different time points is shown in Table 11 and
(94) The anti-tumor effect of 5-FU positive control group (15 mg/kg) was not significant with T/C value and p value of 106% and 0.996, respectively. The low dose (30 mg/kg) and high dose (45 mg/kg) group of LPR-2 had an anti-tumor effect slightly better than that of the 5-FU with tumor volume of 1074 and 827 mm.sup.3, respectively, at the end of experiment, and T/C value of 54% and 42% and p value of 0.177 and 0.089, compared with the physiological saline group. The analysis result of tumor weight was consistent with the tumor volume.
(95) Effect of changes in body weight of tumor-bearing mice in each group is shown in Table 10 and
(96) In summary, in this study, the test drug LPR-2 with dosages of 30 and 45 mg/kg showed a common anti-tumor effect on xenograft tumor model of human hepatoma cell Hep3B, and the animals administrated showed a good tolerance and no death of animals occurred.
Example 8 the Inhibitory Activity of Monomethoxy Polyethylene Glycol (with a Number Average Molecular Weight of 20,000)-Glutamic Acid Dipeptide-Rapamycin Conjugate (LPR-2) and Reference Substance Against H460 Human Non-Small Cell Lung Cancer Cells
(97) (1) Experimental Method and Procedure
(98) (a) Cell Culture
(99) H460 cells were cultured with a monolayer in vitro in RPMI1640 medium supplied with heat-inactivated fetal bovine serum with a volume ratio of 10%, and an incubator at 37 C. with the air containing CO.sub.2 with a proportion of 5%. The tumor cells were passaged with digestion by trypsin-EDTA twice a week. The cells in the exponential growth phase were collected, counted, and used for inoculation.
(100) (b) Inoculation of Tumor Cells, Grouping and Administration
(101) 5.010.sup.6 of H460 tumor cells were suspended in 0.1 ml of PBS, inoculated to each nude mouse at the right shoulder, and there were totally 34 mice inoculated. 8 days later the mean tumor volume was desired to reach about 143 mm.sup.3, the mice with a smaller or larger tumor were removed and the remaining 24 mice were divided into groups randomly according to tumor volume and administrated.
(102) (c) Experimental Scheme
(103) TABLE-US-00013 TABLE 13 The grouping and dosage regimen of experimental animals Compound used Dosage Dosing volume Route of Dosage Group N for treatment (mg/kg) (l/g) administration regimen 1 6 Physiological saline 10 i.v. QW 3W 2 6 Paclitaxel 15 10 i.v. BIW 3W 3 6 LPR-2 30 10 i.v. BIW 3W 4 6 LPR-2 45 15 i.v. QW 3W
(2) Experimental Results
(104) (a) Body Weight
(105) LPR-2 and Paclitaxel had an effect on body weight of mice bearing xenograft tumor model of H460.
(106) Changes in body weight of tumor-bearing mice in each treatment group are shown in Table 14 and
(107) TABLE-US-00014 TABLE 14 The weight of each treatment group at different time points Body weight of animal (g).sup.a LPR-2 LPR-2 Days after Physiological Paclitaxel 30 mg/kg 45 mg/kg inoculation saline 15 mg/kg BIW 3W QW 3W 8 20.6 0.3 20.1 0.4 20.5 0.5 20.2 0.4 12 20.7 0.4 19.9 0.3 20.3 0.5 19.8 0.3 15 21.1 0.4 19.8 0.2 20.7 0.4 20.9 0.3 19 21.4 0.4 19.5 0.2 21.0 0.5 20.7 0.3 22 21.4 0.5 18.6 0.3 20.7 0.4 20.4 0.3 .sup.26.sup.b 22.6 0.5 18.4 0.3 20.9 0.5 21.4 0.3 29 22.8 0.5 18.7 0.4 21.1 0.4 22.1 0.3 Note: .sup.amean value standard error; .sup.blast administration.
(108) (b) Tumor Growth
(109) TABLE-US-00015 TABLE 15 The tumor volume of each treatment group at different time points Tumor volume (mm.sup.3).sup.a LPR-2 LPR-2 Days after Physiological Paclitaxel 30 mg/kg 45 mg/kg inoculation saline 15 mg/kg BIW 3W QW 3W 8 145 21 143 15 143 16 143 18 12 391 66 295 42 213 40 194 33 15 595 112 400 70 208 44 260 46 19 905 149 519 97 259 50 282 46 22 1,176 180 680 110 343 67 346 53 .sup.26.sup.b 1,643 286 944 117 459 70 468 57 29 2,157 387 1,281 184 505 87 549 76 Note: .sup.amean value standard error; .sup.blast administration.
(110) (c) Evaluation of Anti-Tumor Effect
(111) The evaluation indexes of anti-tumor effect of LPR-2 and paclitaxel on xenograft tumor model of H460 are shown in Table 16.
(112) TABLE-US-00016 TABLE 16 Evaluation of anti-tumor effect of each treatment group Tumor volume Tumor weight TGD (mm.sup.3).sup.b RTV.sup.b (mg).sup.b (to 1,000 T/C (%) P value Group N.sup.a 8.sup.th day 29.sup.th day (29.sup.th day) (29.sup.th day) mm.sup.3) RTV TW.sup.c RTV TW.sup.c physiological 6 145 21 2,157 387 15.28 2.40 1602 354 0 100 100 1.000 1.000 saline Paclitaxel 6 143 15 1,281 184 9.03 0.93 1053 146 7 59 66 0.212 0.061 (15 mg/kg) LPR-2 6 143 16 505 87 3.43 0.32 364 54 >9 22 23 0.019 0.000 (30 mg/kg) BIW 3W LPR-2 6 143 18 549 76 3.86 0.42 406 60 >9 25 25 0.022 0.000 (45 mg/kg) QW 3W Note: .sup.athe number of surviving animals in each group after completion of the treatment; .sup.bMean SEM; .sup.cTW (Tumor Weight).
(3) Summary and Discussion of Experimental Results
(113) In the experiment, the pesticide effect in vivo of LPR-2 on subcutaneous transplantation tumor model of H460 human non-small cell lung cancer in nude mice was evaluated. The tumor volume of each treatment group at different time points is shown in Table 15 and
(114) The paclitaxe positive control group of showed a certain anti-tumor effect with a T/C value of 59% and p value of 0.212.
(115) The two groups of LPR-2 with different dosages (30 and 45 mg/kg) showed a significant anti-tumor effect with tumor volume of 505 and 549 mm.sup.3, respectively, at the end of experiment, and T/C value of 22% and 25% and p value of 0.019 and 0.022, compared with the physiological saline group. The analysis result of tumor weight was consistent with the tumor volume.
(116) Effect of changes in body weight of tumor-bearing mice in each group is shown in Table 14 and
(117) In summary, in this study, the test drug LPR-2 with dosages of 30 and 45 mg/kg showed a significant anti-tumor effect on xenograft tumor model of H460 human non-small cell lung cancer, and the animals administrated showed a good tolerance and no death of animals occurred.
Example 9 the Inhibitory Activity of Monomethoxy Polyethylene Glycol (with a Number Average Molecular Weight of 20,000)-Glutamic Acid Dipeptide-Rapamycin Conjugate (LPR-2) and Reference Substance Against Calu-6 Human Lung Cells
(118) (1) Experimental Method and Procedure
(119) (a) Cell Culture
(120) Calu-6 cells were cultured with a monolayer in vitro in MEM medium supplied with heat-inactivated fetal bovine serum with a volume ratio of 10%, and an incubator at 37 C. with the air containing CO.sub.2 with a proportion of 5%. The tumor cells were passaged with digestion by trypsin-EDTA twice a week. The cells in the exponential growth phase were collected, counted, and used for inoculation.
(121) (b) Inoculation of Tumor Cells, Grouping and Administration
(122) 5.010.sup.6 of Calu-6 tumor cells were suspended in 0.1 ml of PBS, inoculated to each nude mouse at the right shoulder, and there were totally 34 mice inoculated. 14 days later the mean tumor volume was desired to reach about 138 mm.sup.3, the mice with a smaller or larger tumor were removed and the remaining 24 mice were divided into groups randomly according to tumor volume and administrated.
(123) (c) Experimental Scheme
(124) TABLE-US-00017 TABLE 17 The grouping and dosage regimen of experimental animals Compound used Dosage Dosing volume Route of Dosage Group N for treatment (mg/kg) (l/g) administration regimen 1 6 Physiological saline 10 i.v. QW 4W 2 6 Paclitaxel 15 10 i.v. BIW 4W 3 6 LPR-2 30 10 i.v. BIW 4W 4 6 LPR-2 45 15 i.v. QW 4W
(2) Experimental Results
(125) (a) Body Weight
(126) LPR-2 and paclitaxel had an effect on body weight of mice bearing xenograft tumor model of Calu-6.
(127) Changes in body weight of tumor-bearing mice in each treatment group are shown in Table 18 and
(128) TABLE-US-00018 TABLE 18 The weight of each treatment group at different time points Body weight of animal (g).sup.a LPR-2 LPR-2 Days after Physiological Paclitaxel 30 mg/kg 45 mg/kg inoculation saline 15 mg/kg BIW 4W QW 4W 14 21.4 0.3 20.7 0.2 20.9 0.4 20.6 0.4 17 21.2 0.2 20.5 0.2 21.0 0.4 20.0 0.4 21 21.3 0.3 20.5 0.3 21.7 0.4 19.9 0.6 24 21.0 0.3 19.6 0.4 21.6 0.3 20.9 0.5 28 21.8 0.3 19.8 0.4 22.4 0.4 21.3 0.6 31 21.6 0.3 19.7 0.6 21.6 0.4 20.7 0.6 35 21.9 0.5 20.1 0.5 22.2 0.4 21.8 0.6 .sup.38.sup.b 22.6 0.5 20.1 0.4 22.3 0.4 21.2 0.5 42 23.6 0.5 20.7 0.4 22.4 0.3 21.5 0.5 Note: .sup.amean value standard error; .sup.blast administration.
(129) (b) Tumor Growth
(130) Changes in tumor volume of each treatment group are shown in Table 19 and
(131) TABLE-US-00019 TABLE 19 The tumor volume of each treatment group at different time points Tumor volume (mm.sup.3).sup.a LPR-2 LPR-2 Days after Physiological Paclitaxel 30 mg/kg 45 mg/kg inoculation saline 15 mg/kg BIW 4W QW 4W 14 138 20 138 14 139 18 137 22 17 196 23 159 18 160 25 152 25 21 301 28 222 23 201 40 204 38 24 407 68 219 26 241 50 245 40 28 568 137 229 37 274 57 293 44 31 769 188 264 53 335 76 323 45 35 1,108 246 281 59 422 108 436 74 .sup.38.sup.b 1,474 325 282 60 504 119 518 87 42 1,889 416 314 72 627 145 720 106 Note: .sup.amean value standard error; .sup.blast administration.
(132) (c) Evaluation of Anti-Tumor Effect
(133) The evaluation indexes of anti-tumor effect of LPR-2 and paclitaxel on xenograft tumor model of calu-6 are shown in Table 24.
(134) TABLE-US-00020 TABLE 20 Evaluation of anti-tumor effect of each treatment group Tumor volume Tumor weight TGD (mm.sup.3).sup.b RTV.sup.b (mg).sup.b (to 1,000 T/C (%) P value Group N.sup.a 14.sup.th day 42.sup.nd day (42.sup.nd day) (42.sup.nd day) mm.sup.3) RTV TW.sup.c RTV TW.sup.c physiological 6 138 20 1,889 416.sup. 14.29 2.45 1616 369 0 100 100 1.000 1.000 saline Paclitaxel 6 138 14 314 72 2.31 0.53 202 52 >8 16 13 0.019 0.054 (15 mg/kg) LPR-2 6 139 18 627 145 4.39 0.59 449 89 >8 31 28 0.042 0.107 (30 mg/kg) BIW 4W LPR-2 6 137 22 720 106 5.41 0.51 602 83 >8 38 37 0.065 0.170 (45 mg/kg) QW 4W Note: .sup.athe number of surviving animals in each group after completion of the treatment; .sup.bMean SEM; .sup.cTW (Tumor Weight).
(3) Summary and Discussion of Experimental Results
(135) In the experiment, the pesticide effect in vivo of LPR-2 on subcutaneous transplantation tumor model of calu-6 human lung cancer cell in nude mice was evaluated. The tumor volume of each treatment group at different time points is shown in Table 19 and
(136) The paclitaxel positive control group showed a significant anti-tumor effect with a T/C value of 16% and p value of 0.019.
(137) The two groups of LPR-2 with different dosages (30 and 45 mg/kg) showed a significant anti-tumor effect with tumor volume of 449 and 602 mm.sup.3, respectively, at the end of experiment, and T/C value of 31% and 38% and p value of 0.042 and 0.065, compared with the physiological saline group.
(138) The analysis result of tumor weight was basically consistent with relative tumor proliferation rate, however, compared with the control group, no significant difference was obtained from the analysis for statistical results of paclitaxel and LPR-2 (30 mg/kg, BIW4W) due to a large difference between tumor weight data in each experimental group.
(139) Effect of changes in body weight of tumor-bearing mice in each group is shown in Table 18 and
(140) In summary, in this study, the test drug LPR-2 with dosages of 30 and 45 mg/kg showed a significant anti-tumor effect on xenograft tumor model of calu-6 human lung cancer cell, and the animals administrated showed a good tolerance and no death of animals occurred.
Example 10 the Inhibitory Activity of Monomethoxy Polyethylene Glycol (with a Number Average Molecular Weight of 20,000)-Glutamic Acid Dipeptide-Rapamycin Conjugate (LPR-2) and Reference Substance Against A549 Human Non-Small Cell Lung Cancer Cells
(141) (1) Experimental Method and Procedure
(142) (a) Cell Culture
(143) A549 cells were cultured with a monolayer in vitro in RPMI1640 medium supplied with heat-inactivated fetal bovine serum with a volume ratio of 10%, and an incubator at 37 C. with the air containing CO.sub.2 with a proportion of 5%. The tumor cells were passaged with digestion by trypsin-EDTA twice a week. The cells in the exponential growth phase were collected, counted, and used for inoculation.
(144) (b) Inoculation of Tumor Cells, Grouping and Administration
(145) 1.010.sup.7 of A549 tumor cells were suspended in 0.1 ml of PBS, inoculated to each nude mouse at the right shoulder, and there were totally 36 mice inoculated. 24 days later the mean tumor volume was desired to reach about 138 mm.sup.3, the mice with a smaller or larger tumor were removed and the remaining 24 mice were divided into groups randomly according to tumor volume and administrated.
(146) (c) Experimental Scheme
(147) TABLE-US-00021 TABLE 21 The grouping and dosage regimen of experimental animals Compound used Dosage Dosing volume Route of Dosage Group N for treatment (mg/kg) (l/g) administration regimen 1 6 Physiological saline 10 i.v. QW 4W 2 6 Paclitaxel 15 10 i.v. BIW 4W 3 6 LPR-2 30 10 i.v. BIW 4W 4 6 LPR-2 45 15 i.v. QW 4W
(2) Experimental Results
(148) (a) Body Weight
(149) LPR-2 and paclitaxel had an effect on body weight of mice bearing xenograft tumor model of A549.
(150) Changes in body weight of tumor-bearing mice in each treatment group are shown in Table 22 and
(151) TABLE-US-00022 TABLE 22 The weight of each treatment group at different time points Body weight of animal (g).sup.a LPR-2 LPR-2 Days after Physiological Paclitaxel 30 mg/kg 45 mg/kg inoculation saline 15 mg/kg BIW 4W QW 4W 24 20.7 0.3 20.6 0.4 21.6 0.5 20.9 0.3 28 21.5 0.3 20.8 0.4 20.9 0.4 20.8 0.1 31 21.3 0.2 20.6 0.4 21.4 0.4 21.1 0.2 35 21.9 0.3 20.8 0.5 21.8 0.4 21.1 0.3 38 22.1 0.3 20.8 0.6 21.6 0.4 21.6 0.3 42 21.9 0.3 21.6 0.6 22.5 0.3 22.0 0.3 45 21.9 0.4 21.3 0.6 22.2 0.3 22.4 0.3 .sup.49.sup.b 22.8 0.4 21.3 0.7 22.0 0.3 21.5 0.3 52 23.0 0.4 21.8 0.6 22.0 0.3 22.1 0.3 Note: .sup.amean value standard error; .sup.blast administration.
(152) (b) Tumor Growth
(153) Changes in tumor volume of each treatment group are shown in Table 23 and
(154) TABLE-US-00023 TABLE 23 The tumor volume of each treatment group at different time points Tumor volume (mm.sup.3).sup.a LPR-2 LPR-2 Days after Physiological Paclitaxel 30 mg/kg 45 mg/kg inoculation saline 15 mg/kg BIW 4W QW 4W 24 138 13 138 13 140 11 139 11 28 259 28 196 24 131 8 115 8 31 345 50 230 35 134 11 126 10 35 521 87 260 49 124 15 122 13 38 654 120 265 48 131 13 120 13 42 849 167 294 41 122 8 124 9 45 1,047 254 327 54 131 9 140 11 .sup.49.sup.b 1,224 251 342 54 128 9 138 14 52 1,391 288 331 49 127 6 145 14 Note: .sup.amean value standard error; .sup.blast administration.
(155) (c) Evaluation of Anti-Tumor Effect
(156) The evaluation indexes of anti-tumor effect of LPR-2 and paclitaxel on xenograft tumor model of A549 are shown in Table 24.
(157) TABLE-US-00024 TABLE 24 Evaluation of anti-tumor effect of each treatment group Tumor volume Tumor weight TGD (mm.sup.3).sup.b RTV.sup.b (mg).sup.b (to 1,000 T/C (%) P value Group N.sup.a 24.sup.th day 52.sup.nd day (52.sup.nd day) (52.sup.nd day) mm.sup.3) RTV TW.sup.c RTV TW.sup.c Physiological 6 138 13 1,391 288 9.81 1.50 1302 326 0 100 100 1.000 1.000 saline Paclitaxel 6 138 13 331 49 2.35 0.16 253 52 >7 24 19 0.019 0.101 (15 mg/kg) LPR-2 6 140 11 127 6 0.94 0.11 102 15 >7 10 8 0.009 0.063 (30 mg/kg) BIW 4W LPR-2 6 139 11 145 14 1.05 0.11 129 20 >7 11 10 0.010 0.069 (45 mg/kg) QW 4W Note: .sup.athe number of surviving animals in each group after completion of the treatment; .sup.bMean SEM; .sup.cTW (Tumor Weight).
(3) Summary and Discussion of Experimental Results
(158) In the experiment, the pesticide effect in vivo of LPR-2 on subcutaneous transplantation tumor model of A549 human non-small cell lung cancer in nude mice was evaluated. The tumor volumes of each treatment group at different time points are shown in Table 23 and
(159) The paclitaxel positive control group showed a significant anti-tumor effect with a T/C value of 24% and p value of 0.019.
(160) The two groups of LPR-2 with different dosages (30 and 45 mg/kg) showed a significant anti-tumor effect with tumor volume of 127 and 145 mm.sup.3, respectively, at the end of experiment, and T/C value of 10% and 11% and p value of 0.009 and 0.010, compared with the physiological saline group.
(161) The analysis result of tumor weight was basically consistent with relative tumor proliferation rate, however, compared with the control group, no significant difference was obtained from the analysis for statistical results of each administration group due to a large difference between tumor weight data in each experimental group.
(162) Effect of changes in body weight of tumor-bearing mice in each group is shown in Table 22 and
(163) In summary, in this study, the test drug LPR-2 with dosages of 30 and 45 mg/kg showed a significant anti-tumor effect on xenograft tumor model of A549 human non-small cell lung cancer, and the animals administrated showed a good tolerance and no death of animals occurred.