Anti-cancer composition consisting of halofuginone and sesquiterpene lactone compounds of artemisia apiacea and use thereof

Abstract

Provided is a combined pharmaceutical composition of HF and an Artemisia annua sesquiterpene lactone compound for treating cancers. The active ingredients of the combined pharmaceutical composition consist of HF and an Artemisia annua sesquiterpene lactone compound. HF and ATS have significant synergistic effect. The activity of the combined pharmaceutical of HF and ATS is comparable or even higher than that of the anti-cancer drug 5-FU.

Claims

1. A combined pharmaceutical composition comprising halofuginone (HF) and an Artemisia annua sesquiterpene lactone compound, wherein active ingredients of said composition consist of HF and the Artemisia annua sesquiterpene lactone compound is artemisinin (ATS).

2. A method of treating colon cancer, the method comprising administering the pharmaceutical composition of claim 1 to a subject.

3. A method of treating breast cancer, the method comprising administering the pharmaceutical composition of claim 1 to a subject.

4. A method of treating liver cancer, the method comprising administering the pharmaceutical composition of claim 1 to a subject.

5. A method of treating gastric cancer, the method comprising administering the pharmaceutical composition of claim 1 to a subject.

6. A method of treating melanoma, the method comprising administering the pharmaceutical composition of claim 1 to a subject.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 illustrates the comparison of the volume of tumor tissues.

(2) FIG. 2 illustrates the HE staining of the animal sections.

(3) FIG. 3 illustrates the comparison of the activity between the combined pharmaceutical of ATS and HF, and the single pharmaceutical of either ATS or HF.

DETAILED DESCRIPTION OF THE INVENTION

Embodiment 1: In Vitro Investigation on the Anti-Cancer Activity of the Combined Pharmaceutical of HF and ATS

(4) Human colon cancer cell line HCT-116 was seeded on a 96-well plate, with 100 μl of cell suspension in each well. The culture medium was DMEM supplemented with 10% FBS and 2 mM glutamate. 24 hours after cell seeding, the 96-well plate was added with HF, ATS, DAT, ASU, ATM, the combination of HF and ATS, the combination of HF and DAT, the combination of HF and ATM, the combination of HF and ASU, respectively, and the culture medium in the control group was not added with any drug. 24 hours after adding the compound, the cell survival rate was assessed using the MTT assay, and the combination index (CI) of the combined pharmaceutical of HF and ATS was calculated using the Calcusyn software. The results are shown in Table 1.
Tumor cell survival rate=the OD value of the experimental group/the OD value of the control group×100%

(5) TABLE-US-00001 TABLE 1 The inhibitory effect of the combined pharmaceutical of HF and ATS on colon cancer cell HCT-116. Cell Combination Cell Combination Cell Combination death Index death Index death Index rate (%) (CI) rate (%) (CI) rate (%) (CI) HF 10 nM 19.41 ± 4.03 HF 20 nM 33.06 ± 3.32 HF 40 nM 59.27 ± 0.41 ATS 35.89 ± 3.91 ATS 320 μM 48.31 ± 3.61 ATS 640 μM 54.42 ± 2.44 160 μM DAT 36.80 ± 3.33 DAT 320 μM 47.32 ± 3.65 DAT 640 μM 55.57 ± 2.43 160 μM ASU 35.15 ± 3.45 ASU 320 μM 48.31 ± 3.69 ASU 640 μM 53.52 ± 2.46 160 μM ATM 33.17 ± 4.46 ATM 320 μM 46.32 ± 3.45 ATM 640 μM 54.50 ± 2.50 160 μM HF 10 nM + 50.90 ± 1.80 0.55 HF 20 nM + 60.15 ± 2.19 0.59 HF 40 nM + 68.76 ± 0.48 0.70 ATS ATS 320 μM ATS 640 μM 160 μM HF 10 nM + 40.90 ± 1.35 0.75 HF 20 nM + 59.12 ± 1.86 0.61 HF 40 nM + 61.24 ± 0.28 0.81 DAT DAT 320 μM DAT 640 μM 160 μM HF 10 nM + 43.91 ± 1.04 0.62 HF 20 nM + 55.49 ± 2.23 0.71 HF 40 nM + 64.71 ± 0.29 0.77 ASU160 μM ASU 320 μM ASU640 μM HF 10 nM + 46.93 ± 1.15 0.59 HF 20 nM + 57.42 ± 2.54 0.70 HF 40 nM + 63.75 ± 1.10 0.78 ATM ATM3 20 μM ATM 640 μM 160 μM

(6) Breast cancer cell line MCF-7 was seeded on a 96-well plate, with 100 μl of cell suspension in each well. The culture medium was DMEM supplemented with 10% FBS and 2 mM glutamate. 24 hours after cell seeding, the 96-well plate was added with HF, ATS, DAT, ASU, ATM, the combination of HF and ATS, the combination of HF and DAT, the combination of HF and ATM, and the combination of HF and ASU, respectively, and the culture medium of the control group was not added with any drug. 24 hours after adding the compound, the cell survival rate was assessed using the MTT assay, and the combination index (CI) of the combined pharmaceutical of HF and ATS was calculated using the Calcusyn software. The results are shown in Table 2.

(7) TABLE-US-00002 TABLE 2 The inhibitory effect of the combined pharmaceutical of HF and ATS on breast cancer cell MCF-7. Cell Cell death Combination death Combination Cell Combination rate Index rate Index death Index (%) (CI) (%) (CI) rate (%) (CI) HF  2.00 ± 1.28 HF 20 nM  3.00 ± 0.55 HF 40 nM  9.10 ± 1.79 10 nM ATS  9.50 ± 0.77 ATS 320 μM 16.50 ± 1.47 ATS 640 μM 33.90 ± 2.00 160 μM DAT  8.81 ± 0.83 DAT 320 μM 16.32 ± 1.65 DAT 640 μM 32.59 ± 2.43 160 μM ASU  9.15 ± 0.92 ASU 320 μM 16.31 ± 1.69 ASU 640 μM 33.57 ± 2.56 160 μM HF 15.80 ± 0.37 0.86 HF 20 nM + 24.30 ± 3.01 0.71 HF 40 nM + 44.70 ± 2.40 0.37 10 nM + ATS 320 μM ATS 640 μM ATS 160 μM HF10 nM + 15.92 ± 0.64 0.84 HF 20 nM + 22.12 ± 1.85 0.77 HF 40 nM + 38.24 ± 0.28 0.71 DAT DAT 320 μM DAT 640 μM 160 μM HF 13.92 ± 1.06 0.91 HF 20 nM + 20.49 ± 2.23 0.76 HF 40 nM + 37.71 ± 0.29 0.71 10 nM + ASU 320 μM ASU 640 μM ASU 160 μM HF 14.94 ± 1.13 0.89 HF 20 nM + 22.42 ± 2.22 0.77 HF 40 nM + 35.75 ± 1.18 0.77 10 nM + ATM ATM640 μM ATM160 μM 320 μM

(8) Liver cancer cell line HepG2 was seeded on a 96-well plate, with 100 μl of cell suspension in each well. The culture medium was DMEM supplemented with 10% FBS and 2 mM glutamate. 24 hours after cell seeding, the 96-well plate was added with HF, ATS, DAT, ASU, ATM, the combination of HF and ATS, the combination of HF and DAT, the combination of HF and ATM, and the combination of HF and ASU, respectively, and the culture medium of the control group was not added with any drug. 24 hours after adding the compound, the cell survival rate was assessed using the MTT assay, and the combination index (CI) of the combined pharmaceutical of HF and ATS was calculated using the Calcusyn software. The results are shown in Table 3.

(9) TABLE-US-00003 TABLE 3 The inhibitory effect of the combined pharmaceutical of HF and ATS on liver cancer cell HepG2. Cell death Combination Combination Combination rate Index Cell death Index Cell death Index (%) (CI) rate (%) (CI) rate (%) (CI) HF 0.70 ± 3.11 HF 20 nM 1.00 ± 2.06 HF 40 nM 4.00 ± 2.21 10 nM ATS 0.90 ± 0.95 ATS 1.60 ± 0.08 ATS 640 μM 6.40 ± 2.71 160 μM 320 μM DAT 0.81 ± 0.83 DAT 1.72 ± 0.15 DAT 640 μM 6.59 ± 2.58 160 μM 320 μM ASU 0.95 ± 0.72 ASU 1.86 ± 0.09 ASU 640 μM 6.57 ± 2.06 160 μM 320 μM HF 7.90 ± 4.30 0.18 HF 20 nM + 13.20 ± 5.67  0.16 HF 40 nM + 34.10 ± 4.63  0.08 10 nM + ATS ATS 640 μM ATS 320 μM 160 μM HF10 nM + 2.90 ± 0.31 0.45 HF 20 nM + 4.12 ± 0.81 0.41 HF 40 nM + 11.24 ± 0.28  0.22 DAT DAT DAT 640 μM 160 μM 320 μM HF 1.91 ± 0.04 0.62 HF 20 nM + 3.10 ± 0.41 0.55 HF 40 nM + 7.21 ± 0.29 0.48 10 nM + ASU ASU 640 μM ASU 320 μM 160 μM HF 1.94 ± 1.18 0.67 HF 20 nM + 5.42 ± 2.22 0.38 HF 40 nM + 8.75 ± 1.18 0.39 10 nM + ATM ATM 640 μM ATM 320 μM 160 μM

(10) Gastric cancer cell line MGC803 was seeded on a 96-well plate, with 100 μl of cell suspension in each well. The culture medium was DMEM supplemented with 10% FBS and 2 mM glutamate. 24 hours after cell seeding, the 96-well plate was added with HF, ATS, DAT, ASU, ATM, the combination of HF and ATS, the combination of HF and DAT, the combination of HF and ATM, and the combination of HF and ASU, respectively, and the culture medium of the control group was not added with any drug. 24 hours after adding the compound, the cell survival rate was assessed using the MTT assay, and the combination index (CI) of the combined pharmaceutical of HF and ATS was calculated using the Calcusyn software. The results are shown in Table 4.

(11) TABLE-US-00004 TABLE 4 The inhibitory effect of the combined pharmaceutical of HF and ATS on gastric cancer cell MGC803. Cell Combination Cell Combination Combination death Index death Index Cell death Index rate (%) (CI) rate (%) (CI) rate (%) (CI) HF  8.10 ± 2.77 HF 20.00 ± 4.18 HF 40 nM 29.00 ± 3.31 10 nM 20 nM ATS  1.00 ± 3.31 ATS  4.60 ± 5.51 ATS 40.00 ± 3.18 160 μM 320 μM 640 μM DAT  1.81 ± 0.83 DAT  5.32 ± 1.65 DAT 42.59 ± 2.43 160 μM 320 μM 640 μM ASU  1.15 ± 0.92 ASU  5.31 ± 1.69 ASU 43.57 ± 2.56 160 μM 320 μM 640 μM HF 23.78 ± 4.73 0.42 HF 34.54 ± 4.92 0.46 HF 40 nM + 64.70 ± 5.46 0.20 10 nM + 20 nM + ATS ATS ATS 640 μM 160 μM 320 μM HF10 nM + 20.90 ± 1.33 0.75 HF 29.16 ± 3.56 0.52 HF 40 nM + 61.24 ± 0.28 0.38 DAT 20 nM + DAT 160 μM DAT 640 μM 320 μM HF 23.21 ± 3.14 0.62 HF 27.32 ± 2.26 0.59 HF 40 nM + 64.71 ± 3.29 0.21 10 nM + 20 nM + ASU ASU ASU 640 μM 160 μM 320 μM HF 20.94 ± 1.55 0.79 HF 28.42 ± 2.25 0.57 HF 40 nM + 60.75 ± 1.16 0.41 10 nM + 20 nM + ATM ATM ATM 640 μM 160 μM 320 μM

(12) Melanoma cell line A375 was seeded on a 96-well plate, with 100 μl of cell suspension in each well. The culture medium was DMEM supplemented with 10% FBS and 2 mM glutamate. 24 hours after cell seeding, the 96-well plate was added with HF, ATS, DAT, ASU, ATM, the combination of HF and ATS, the combination of HF and DAT, the combination of HF and ATM, and the combination of HF and ASU, respectively, and the culture medium of the control group was not added with any drug. 24 hours after adding the compound, the cell survival rate was assessed using the MTT assay, and the combination index (CI) of the combined pharmaceutical of HF and ATS was calculated using the Calcusyn software. The results are shown in Table 5.

(13) TABLE-US-00005 TABLE 5 The inhibitory effect of the combined pharmaceutical of HF and ATS on melanoma cell A375. Cell death Combination Cell Combination Cell Combination rate Index death Index death Index (%) (CI) rate (%) (CI) rate (%) (CI) HF  5.70 ± 5.44 HF 20 nM 17.70 ± 2.82 HF 40 nM 35.70 ± 4.76 10 nM ATS 16.50 ± 4.30 ATS 320 μM 26.30 ± 1.33 ATS 640 μM 46.50 ± 3.57 160 μM DAT 16.81 ± 5.83 DAT 320 μM 26.35 ± 1.64 DAT 640 μM 42.55 ± 4.42 160 μM ASU 16.15 ± 4.90 ASU 320 μM 25.38 ± 1.65 ASU 640 μM 47.59 ± 3.93 160 μM HF 33.70 ± 4.75 0.42 HF 20 nM + 54.40 ± 4.33 0.21 HF 40 nM + 66.60 ± 0.94 0.20 10 nM + ATS 320 μM ATS 640 μM ATS 160 μM HF10 nM + 27.90 ± 1.35 0.74 HF 20 nM + 47.13 ± 1.86 0.46 HF 40 nM + 51.24 ± 0.28 0.31 DAT DAT 320 μM DAT 640 μM 160 μM HF 23.95 ± 1.23 0.62 HF 20 nM + 35.49 ± 2.23 0.80 HF 40 nM + 44.71 ± 0.29 0.49 10 nM + ASU 320 μM ASU640 μM ASU 160 μM HF 24.95 ± 1.08 0.89 HF 20 nM + 42.51 ± 1.20 0.57 HF 40 nM + 45.75 ± 0.18 0.42 10 nM + ATM 320 μM ATM 640 μM ATM 160 μM

(14) In reference to the method in the Soriano A F et al., Synergistic effects of new chemopreventive agents and conventional cytotoxic agents against human lung cancer cell lines, Cancer Res, 1999, 59 (24): 6178-6184, the inhibitory effect investigation on colon cancer cell HCT-116 shows that the CIs of the combination of HF and ATS were was 0.55-0.7. Such synergistic effect exhibited a decreasing trend with the increase of the pharmaceutical concentration, regarded as moderate synergistic effect. The CIs of the combination of HF and DAT were 0.75, 0.61, and 0.81 respectively, regarded as moderate and low synergistic effect. The CIs of the combination of HF and ASU were 0.62-0.77. Such synergistic effect exhibited a decreasing trend with the increase of the pharmaceutical concentration, regarded as moderate synergistic effect. The CIs of the combination of HF and ATM were 0.50-0.78, regarded as moderate synergistic effect. The inhibitory effect investigation on breast cancer cell MCF-7 shows that the CIs of the combination of HF and ATS were 0.37-0.86. Such synergistic effect exhibited an increasing trend with the increase of the pharmaceutical concentration. The CIs of the combination of HF and DAT were 0.84, 0.77, and 0.71 respectively, regarded as low synergistic effect. The CIs of the combination of HF and ASU were 0.91, 0.76, and 0.71, respectively, having synergistic effect. The CIs of the combination of HF and ATM were 0.88, 0.77, and 0.77 respectively, having moderate synergistic effect. The inhibitory effect investigation on liver cancer cell HepG2 shows that the CIs of combination of HF and ATS were 0.08-0.18. Such synergistic effect exhibited an increasing trend with the increase of the pharmaceutical concentration. The combined pharmaceutical of HF and artemisinin can be regarded as strong synergistic effect. The CIs of the combination of HF and DAT were 0.45-0.22, regarded as high and strong synergistic effect. The CIs of the combination of HF and ASU were 0.62, 0.55, and 0.48, respectively, regarded as high synergy. The CIs of the combination of HF and ATM were 0.67, 0.38, and 0.39, respectively, having moderate synergistic effect. The inhibitory effect investigation on gastric cancer cell MGC803 shows that the CIs of the combination of HF and ATS were 0.20-0.42. Such synergistic effect exhibited an increasing trend with the increase of the pharmaceutical concentration. The combined pharmaceutical of HF and ATS can be regarded as high synergistic effect. The CIs of the combination of HF and DAT were 0.75-0.38. Such synergistic effect exhibited an increasing trend with the increase of the pharmaceutical concentration, regarded as moderate and high synergistic effect. The CIs of the combination of HF and ASU 0.62-0.21, the synergistic effect of which exhibited an increasing trend with the increase of the pharmaceutical concentration, regarded as moderate and high synergistic effect, and can be understood as high and strong synergy. The CIs of the combination of HF and ATM were 0.79, 0.57, and 0.41, respectively, regarded as moderate synergistic effect. The inhibitory effect investigation on melanoma cell A375 shows that the CIs of the combination of HF and ATS were 0.20-0.42. Such synergistic effect exhibited an increasing trend with the increase of the pharmaceutical concentration. The combined pharmaceutical of HF and ATS can be regarded as high synergistic effect. The CIs of the combination of HF and DAT were 0.74-0.31, the synergistic effect of which exhibited an increasing trend with the increase of the pharmaceutical concentration, regarded as moderate and low synergistic effect. The CIs of the combination of HF and ASU were 0.62, 0.80, and 0.21, respectively, regarded as moderate and low synergistic effect. The CIs of the combination of HF and ATM were 0.89, 0.57, and 0.42, respectively, having synergistic effect.

(15) It can be seen from the aforementioned inhibitory results of the combined pharmaceutical of HF and an Artemisia annua sesquiterpene lactone compound on the 5 cancer cell lines, that the activity of the combined pharmaceutical of HF and ATS and derivatives thereof is significantly higher than that of the HF alone and Artemisia annua sesquiterpene lactone compounds alone. Wherein, the activity of the combined pharmaceutical of ATS and HF is the most significant.

Embodiment 2: In Vivo Investigation on the Inhibitory Effect of the Combinational Pharmaceutical of HF and ATS on Colon Cancer

(16) The combination of ATS and HF, ATS alone and HF alone were subjected to animal experiments, with 5-FU as a control drug. The colon cancer cells HCT-116 were inoculated under the skin of female nude mice. Each mouse was inoculated with 1×10.sup.6 cells. Pharmaceuticals were administrated after 5 days when the tumor volume reached about 100 mm.sup.3. The dose of HF was 5 μg/kg, the dose of ATS was 50 mg/kg, the dose of the combined pharmaceutical of HF and ATS was the sum of the doses of HF and ATS, and the dose of 5-FU was 10 mg/kg. Each group was administered intraperitoneally once a day. After 15 days, the tumors were excised (FIG. 1), and were subjected to pathological biopsy examination (FIG. 2). It was found that the combination of HF and ATS had significant synergistic effect, which was significantly better than ATS or HF alone (FIG. 3). Moreover, the activity of the combination of HF and ATS was higher than that of 5-FU.

(17) The combination of HF and ATS with different mass ratios were subjected to animal experiments according to the aforementioned method, wherein the first group was administered with 100 μg/kg HF and 20 mg/kg ATS, and the second group was administered with 2 μg/kg HF and 100 mg/kg ATS. Results show that the activities of the combinations of ATS and HF with different mass ratios were comparable with the efficacy of 5-FU. The efficacies of the combinations of ATS and HF with different mass ratios were still higher than that of ATS or HF alone.

(18) In summary, the combination of ATS and HF has significant therapeutic effect on colon cancer. The activity of the combination of ATS and HF is comparable with the efficacy of 5-FU. Within the range of 0.1:20 to 0.002:100 of HF and ATS (the mass ratio of HF:ATS is 1:2×10.sup.2-5×10.sup.5), the HF and ATS has good synergistic effect, and can be used as an effective combined pharmaceutical.