Medicine for combined use in cancer treatment

Abstract

A medicine for combined use in cancer treatment, comprising chlorogenic acid and a hormone drug which have unit preparations of the same or different specifications and which are administered either together or separately, as well as comprising a pharmaceutically acceptable carrier. The combined use of the chlorogenic acid and hormone drug achieves the effects of synergistic interaction, which thus overcome the defects of single drug treatment, such as a major toxic side effect and a poor treatment outcome and may reverse the drug resistance to hormone drugs and has good prospects for clinical application.

Claims

1. A combined medicine for treating cancer, comprising chlorogenic acid and a hormone drug in a unit preparation comprising the same or different dosage forms, and a pharmaceutically acceptable carrier, wherein said hormone drug is selected from fulvestrant, abarelix, degarelix, and combinations thereof, chlorogenic acid is administrated at 10-40 mg/kg, fulvestrant is administrated at 40 mg/kg, abarelix is administrated at 15 mg/kg, and degarelix is administrated at 30 mg/kg, and said cancer is hormone-dependent breast, prostate, ovarian, and/or cervical cancer(s).

2. The combined medicine according to claim 1, wherein a weight ratio of said chlorogenic acid and the hormone drug is 2:3-1:3, and the hormone drug is fulvestrant or degarelix.

3. The combined medicine according to claim 1, wherein said unit preparation is an injectable or oral formulation of drug.

Description

EXAMPLES

Example 1 In Vivo Animal Experiments on the Combination of Chlorogenic Acid and Fulvestrant for Treating Breast Cancer in Mice

(1) 1. Material

(2) Test drug: chlorogenic acid, fulvestrant.

(3) Test cell lines: EMT-6 mouse breast cancer cell lines.

(4) Test animals: BALB/C-nu mice, ♀, weight 16-21 g.

(5) 2. Experimental Method.

(6) Cells in exponential phase of growth were taken out, digested with trypsin to remove the cell wall, and then cell suspension was prepared by addition of saline. The prepared cell suspension was inoculated under the left anterior armpit of mice at 0.2 ml/mouse (about 1×10.sup.6 cells), and the mice were randomly divided into four groups as weight, i.e. fulvestrant group, chlorogenic acid group, fulvestrant+chlorogenic acid group, and the negative control group, respectively, each group having 6 mice.

(7) Fulvestrant group received drug once by intraperitoneal injection on the next day after inoculation; chlorogenic acid group received drug once every day by intraperitoneal injection from the next day after inoculation, and the drug was successively administrated 15 times; combined medicine group received intraperitoneal injection of fulvestrant once on the next day after inoculation, and intraperitoneal injection of chlorogenic acid once every day from the next day after inoculation; the negative control group received saline by intraperitoneal injection once every day from the next day after inoculation, and saline was continuously given 15 times. When the tumor volume in the negative group was about 0.5 cm.sup.3, the experiment was stopped. The mice were sacrificed by cervical dislocation and weighed. The tumors were removed and weighed to calculate the tumor suppression rate.

(8) 3. Data Processing

(9) (1) tumor inhibition rate %=[1−(the average tumor weight in the test group/the average tumor weight in the negative group)]×100%

(10) (2) If both drugs are combined, Q=E(a+b)/(Ea+Eb−Ea×Eb), in which E(a+b) is the inhibition rate for the combination of two drugs, i.e. the tested combined effect; Ea and Eb is respectively the inhibition rates of two drugs when they are separately used; the denominator (Ea+Eb−Ea×Eb) is the expected combined effect. When Q value is 0.85-1.15, the combined effect of both drugs is added up (+); When Q value is 1.15-20, the combined effect of both drugs is synergistic (++); When Q value is >20, the synergistic effect is obvious (+++); When Q value is 0.05-0.85, the combined effect of both drugs is antagonistic; when Q value is <0.05, the antagonistic effect is obvious.

(11) 4. Experimental Results

(12) For the effect of the combination of chlorogenic acid and fulvestrant on mouse xenograft tumor inhibition, the results are shown in Table 1.

(13) TABLE-US-00001 TABLE 1 Effect of the combined drug on inhibition of mouse xenograft tumor of EMT-6 breast cancer. Tumor Dosage Tumor inhibitory Q Groups (mg .Math. kg.sup.−1) weight (g) rate (%) value chlorogenic 10 0.897 ± 0.346* 31.94 — acid group 20 0.831 ± 0.221* 36.95 — 40 0.764 ± 0.539* 42.03 — The Fulvestrant 40 +  0.267 ± 0.174** 79.74 1.297 combined chlorogenic acid 10 drug group Fulvestrant 40 +  0.209 ± 0.352** 84.14 1.308 chlorogenic acid 20 Fulvestrant 40 +  0.228 ± 0.209** 82.70 1.231 chlorogenic acid 40 Fulvestrant 40 0.746 ± 0.311* 43.40 — group Negative N.S 1.318 ± 0.731  — — control group Compared with the negative control group, *p < 0.05, **p < 0.01.

(14) For the combined use of 40 mg/kg fulvestrant with 10 mg/kg, 20 mg/kg, 40 mg/kg chlorogenic acid, Q values for inhibition of mouse xenograft tumor of EMT-6 breast cancer are 1.231-1.308 and locate in the range of 1.15-20, and thus both drugs have synergistic effect.

Example 2 In Vivo Animal Experiments on the Combination of Chlorogenic Acid and Barelix or Degarelix for Treating Prostate Cancer in Mice

(15) 1. Material

(16) Test drug: chlorogenic acid, abarelix, degarelix.

(17) Test cell lines: RM-1 mouse prostate cancer cell lines.

(18) Test animals: Kunming mice, ♂, weight 17-24 g.

(19) 2. Experimental Method

(20) Cells in exponential phase of growth were taken out, digested with trypsin to remove the cell wall, and then cell suspension was prepared by addition of saline. The prepared cell suspension was inoculated under the left anterior armpit of mice at 0.2 ml/mouse (about 1×10.sup.6 cells), and the mice were randomly divided into six groups as weight, i.e. abarelix group, degarelix group, chlorogenic acid group, abarelix+chlorogenic acid group, degarelix+chlorogenic acid group, and the negative control group, respectively, each group having 6 mice.

(21) Abarelix group received drug once by intraperitoneal injection on the next day after inoculation; degarelix group received drug once by subcutaneous injection on the next day after inoculation; chlorogenic acid group received drug once every day by intraperitoneal injection from the next day after inoculation, and the drug was successively administrated 15 times; abarelix+chlorogenic acid group received abarelix once by intraperitoneal injection on the next day after inoculation, and received chlorogenic acid once every day by intraperitoneal injection from the next day after inoculation, and chlorogenic acid was administrated 15 times; degarelix+chlorogenic acid group received degarelix once by subcutaneous injection on the next day after inoculation, and received chlorogenic acid once every day by intraperitoneal injection from the next day after inoculation, and chlorogenic acid was administrated 15 times; the negative control group received saline by intraperitoneal injection once every day from the next day after inoculation, and saline was continuously given 15 times. When the tumor volume in the negative group was about 0.5 cm.sup.3, the experiment was stopped. The mice were sacrificed by cervical dislocation and weighed. The tumors were removed and weighed to calculate the tumor inhibitory rate.

(22) 3. Data Processing

(23) The data processing is same to that in example 1.

(24) 4. Experimental Results

(25) For the effect of the combination of chlorogenic acid and abarelix or degarelix on mouse xenograft tumor inhibition, the results are shown in Table 2.

(26) TABLE-US-00002 TABLE 2 Effect of the combined drug on inhibition of mouse xenograft tumor of prostate cancer. Tumor Dosage Tumor inhibitory Q Groups (mg .Math. kg.sup.−1) weight (g) rate (%) value chlorogenic 10 1.175 ± 0.117  24.05 — acid group 20 1.116 ± 0.358  27.86 — 40 1.105 ± 0.641  28.57 — abarelix + abarelix15 + 0.275 ± 0.419** 82.22 1.455 chlorogenic chlorogenic acid10 acid group abarelix15 + 0.241 ± 0.303** 84.42 1.439 chlorogenic acid20 abarelix15 + 0.264 ± 0.16**1 82.93 1.404 chlorogenic acid40 degarelix + degarelix30 + 0.238 ± 0.224** 84.62 1.354 chlorogenic chlorogenic acid10 acid group degarelix30 + 0.204 ± 0.193** 86.81 1.349 chlorogenic acid20 degarelix30 + 0.211 ± 0.462** 86.36 1.334 chlorogenic acid20 Abarelix 15 0.886 ± 0.521*  42.73 — group Degarelix 30 0.764 ± 0.218*  50.61 — group Negative N.S 1.547 ± 0.258  — — control group Compared with the negative control, *p < 0.05, **p < 0.01.

(27) For the combined use of 15 mg/kg abarelix with 10 mg/kg, 20 mg/kg, 40 mg/kg chlorogenic acid, Q values for inhibition of mouse xenograft tumor of RM-1 prostate cancer are 1.334-1.354 and locate in the range of 1.15-20, indicating both drugs have synergistic effect.

(28) For the combined use of 30 mg/kg degarelix with 10 mg/kg, 20 mg/kg, 40 mg/kg chlorogenic acid,

(29) Q values for inhibition of mouse xenograft tumor of RM-1 prostate cancer are 1.334-1.354 and locate in the range of 1.15-20, indicating both drugs have synergistic effect.

Example 3 Animal Experiments on the Combination of Chlorogenic Acid and Abarelix Against Multi-Drug Resistant Cancers

(30) 1. Material

(31) Test drug: chlorogenic acid, abarelix.

(32) Test cell lines: RM-1 mouse prostate cancer cell lines. RM-1 cell lines were induced by increasing concentration gradient of aberlix and established by clone and screen. Prior to experiment, cell lines were cultured without drug.

(33) Test animals: Kunming mice, ♂, weight 17-24 g.

(34) 2. Experimental Method

(35) 2.1 Establishment of Experimental Animal Tumor Model

(36) Drug-resistant cell lines after removal of drug were adjusted to the concentration of 1×10.sup.7/mL with culture medium, and inoculated under the right anterior armpit of mice at 0.1 ml/mouse.

(37) 2.2 Method of Administration

(38) After the mean diameter of tumor reached 100 mm.sup.3, mice were divided into 4 groups, i.e. abarelix group, chlorogenic acid+abarelix group, chlorogenic acid group, and the negative control group, respectively.

(39) Abarelix group: intraperitoneal injection, once every seven days, 15 mg/kg, two doses in total.

(40) chlorogenic acid+abarelix group: chlorogenic acid was intraperitoneally injected, once every day, 10 mg/kg, continuous administration for 5 days; After withdrawal of chlorogenic acid, abarelix was intraperitoneally injected on the next day, once every 7 days, 15 mg/kg, two doses in total.

(41) Chlorogenic acid group: intraperitoneal injection, once every day, 10 mg/kg, continuous administration for 5 days; after that, saline was intraperitoneally injected, once every day, continuing 10 days.

(42) Negative control group: intraperitoneal injection of saline, once every day, continuing 15 days.

(43) 2.3 Evaluation of Anti-Tumor Effect

(44) After completion of administration, the experiment was stopped, and mice were sacrificed by cervical dislocation and weighed. The tumors were removed and weighed to calculate the tumor inhibitory rate.

(45) 3. Data Processing

(46) tumor inhibition rate %=[1−(the average tumor weight in the test group/the average tumor weight in the negative group)]×100%

(47) 4. Experimental Results

(48) For the effect on tumor inhibition rate against drug-resistant xenograft tumors in each test group, the results are shown in Table 3.

(49) TABLE-US-00003 TABLE 3 Effect on the tumor weight and the tumor inhibitory rate of mouse xenograft tumor of drug-resistant RM-1 prostate cancer (x ± s) Tumor Animal inhib- Dosage number Tumor itory Groups (mg .Math. kg.sup.−1) (n) weight (g) rate (%) chlorogenic 10 8 2.251 ± 0.581 8.41 acid abarelix 15 8 2.339 ± 0.614 4.84 chlorogenic chlorogenic 8   .sup. 0.624 ± 0.632**ΔΔ 74.54 acid + acid10 + abarelix abarelix15 Negative N.S 8 2.458 ± 0.874 — control group Compared with the negative control, *p < 0.05, **p < 0.01; compared with nilotinib, Δp < 0.05, ΔΔp < 0.01

(50) Results indicated that in chlorogenic acid group and in abarelix group, the inhibitory rate of mouse xenograft tumor of drug-resistant RM-1 prostate cancer was weaker, without obvious suppression effect. While in chlorogenic acid+abarelix group, the inhibitory rate of mouse xenograft tumor of drug-resistant RM-1 prostate cancer was significant, indicating that chlorogenic acid can effectively solve the drug-resistance of resistant RM-1 prostate cancer.

(51) In summary, the combination of chlorogenic acid and hormone drugs can be used for treatment of breast, prostate, ovarian or cervical cancers, and in the combination, the ratio of chlorogenic acid and hormone drugs is 8:3-1:4, preferably 2:3-1:3. Compared with the therapeutic effect of single drug, the combination of chlorogenic acid and hormone drugs realizes the synergistic effect, and the therapeutic effect is better. Moreover, the combination can reverse the drug resistance of hormone drugs, and have good clinical application prospects.