NL2020118B1 - Use of Apiole derivatives for manufacture of a medicant for the treatment of anticancer drugs - Google Patents

Abstract

A use of Apiole derivatives for manufacture of a medicant for the treatment of anticancer drugs provided in the present disclosure includes AP—02, AP—04 and AP—05; an Apiole deriva— tive is characteristic of the effective dose between 0.016 and 9.56 mg/kg each time, the better dose between 0.041 and 4.88 mg/kg each time, and the optimal dose between 0.081 and 2.44mg/kg each time; the Apiole derivative contributes to apoptosis and the over expression of the tumor suppressor pro— tein p53 for the effect of inhibiting growth of tumors.

Description

Use of Apiole derivatives for manufacture of a medicant for the treatment of anticancer drugs

BACKGROUND OF THE INVENTION 1. Field of the invention

The present invention relates to a use of Apiole derivatives for manufacture of a medicant for the treatment of anticancer drugs, particularly active substances inhibiting proliferations of cancer cells for the anticancer effect. 2. Description of the prior art

Apiole, also known as apiol and presented in the chemical formula of 2,5-dimethoxy safrole, is colorless acicular crystals (hydrocarbon oxide solids) with faint aroma found by Heinrich Christoph Link, a pharmacist who distilled oil with steam at Leipzig, Germany, in 1715, and considered as an effective emmenagogue in 1855. Apiole and dillapiol (C12H14O4; molecular weight=222.24) have similar or common pharmacologic actions such as antisepsis, insect repellence and sedation. Natural Apiole can be found in various plants, for example, Petroselinum crispum, Apium graveolens, Oenanthe aquatica, Oenanthe crocata, Anethum graveolens, Foeniculum vulgare, Peperomia pellucida, Piper cubeba, Levisticum officinale, Sassafras albidum, etc.

In recent years, more and more Chinese died of cancers and the death toll attributed to cancers accounted for 26.8% of the total death toll according to reports of the 2012 Chinese Urban Mortality and Data. Accordingly, as the leading cause of death, cancers have drawn more attentions in their prevention, control and treatment gradually.

In virtue of medical effects of Apiole arising wide concerns of people, the Taiwanese patent applicant presented the "Medical applications of Apiole to treat colorectal cancers" (Taiwan Patent Application No. TW 201141474) in which pharmaceutical compositions, cell cycle inhibitors and colorectal cancer cytotoxic agents are disclosed. However, the effects of anticancer compositions in Apiole derivatives were not studied sufficiently. In the U.S. Patent of "Derivatives of Apiole and related monolignans and use thereof" (U.S. Publication No. 20130012477), AP-02, a derivative of Apiole, is used in inhibiting cytochrome P450 and taken as a pesticide and AP-04, another derivative of Apiole , is an intermediate product during syntheses of compounds but not explained in detail.

The anticancer effect of Apiole derivatives is still unknown to people in spite of Apiole performing well in inhibiting colorectal cancers as shown in evidences. Considering the effect of Apiole to inhibit colorectal cancers, the patent applicant, who studied and analyzed the anticancer effects of various Apiole derivatives in years, presents the patent application of "Apiole derivatives with medical applications for preparation of anticancer drugs".

SUMMARY OF THE INVENTION

The present invention is to provide a use of Apiole derivatives for manufacture of a medicant for the treatment of anticancer drugs .

According to the invention, wherein the Apiole derivative has the chemical structural formula as follows:

wherein Ri or R2 can be a hydrogen atom, an alkyl with one to three carbon atoms, an alkenyl with one to three carbon atoms, an alkynyl with one to three carbon atoms or an ether with one to three carbon atoms.

According to the invention, wherein the Apiole derivative is AP-02, which is 5-Allyl-6-methoxybenzo[d][1,3]dioxole, as shown in the following chemical structural formula:

According to the invention, wherein the Apiole derivative is AP-04, which is 5-methoxybenzo[d][1,3]dioxole, as shown in the following chemical structural formula:

According to the invention, wherein the Apiole derivative is AP-05, which is 5-ally benzo[d][1,3]dioxole, as shown in the following chemical structural formula:

According to the invention, wherein the Apiole derivative is characteristic of the effective dose between 0.016 and 9.56 mg/kg each time.

According to the invention, wherein the Apiole derivative is characteristic of the better dose between 0.041 and 4.88 mg/kg each time.

According to the invention, wherein the Apiole derivative is characteristic of the optimal dose between 0.081 and 2.44mg/kg each time.

According to the invention,, wherein the Apiole derivative is able to inhibit proliferations of hepatoma cells, colon carcinoma cells, lung carcinoma cells and breast carcinoma cells .

According to the invention, wherein the Apiole derivative contributes to apoptosis for the effect of inhibiting growth of tumors.

According to the invention, wherein the Apiole derivative contributes to the over expression of the tumor suppressor protein p53 for the effect of inhibiting growth of tumors.

According to the invention, wherein the Apiole derivative can be a pharmaceutically acceptable carrier, excipient or diluting agent.

According to the invention,, wherein the Apiole derivative has the dosage form selected from a combination of a solution, a suspension liquid, an emulsion, powders, a pastille, a pill, syrups, a buccal tablet, a tablet, a chewing tablet, stiff pastes and a capsule.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1A illustrates the results of the MTT assay for the breast carcinoma cell line, MDA-MB-231; FIG. IB illustrates the results of the MTT assay for the breast carcinoma cell line, ZR75; FIG. 1C illustrates the results of the MTT assay for the normal breast cell line, MCF-10A; FIG. 2A illustrates the results of the MTT assay for the lung carcinoma cell line, A549; FIG. 2B illustrates the results of the MTT assay for the lung carcinoma cell line, PE089; FIG. 20 illustrates the results of the MTT assay for the normal lung cell line, HEL-299; FIG. 3A illustrates the results of the MTT assay for the hepatoma cell line, Hep3B; FIG. 3B illustrates the results of the MTT assay for the hepatoma cell line, HepG2; FIG. 3C illustrates the results of the MTT assay for the normal liver cell line, BNL cl. 2; FIG. 4A illustrates the results of the MTT assay for the colon carcinoma cell line, Colon 205; FIG. 4B illustrates the results of the MTT assay for the colon carcinoma cell line, HT-29; FIG. 4C illustrates the results of the MTT assay for the normal colon cell line, FHC; FIG. 5 illustrates the capacities of AP-02 with different doses to kill off cancer cells; FIGS.6A illustrate the analytic results for the cell cycle and 6B and the induced apoptosis; FIG. 7A illustrates the effect of AP-02 to inhibit growth of tumors; FIG. 7B illustrates the analytic results for reduced weights of nude mice; and FIG. 8 illustrates the analytic results for protein expressions of mice's tumor cells.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The Apiole derivatives with medical applications for preparation of anticancer drugs are explained in but not limited to following embodiments. Unless otherwise specified, all materials in the present invention are available in the market. Embodiment 1: Evaluations of effects of three anticancer drugs to inhibit growth of tumors in vitro (cell experiments)

There are four anticancer drugs in the present invention, Apiole and Apiole derivatives including AP-02, AP-04 and AP-05 wherein AP-02, AP-04 and AP-05 are shown as follows:

The cell lines adopted in experiments include two colon carcinoma cell lines and one normal colon cell line, two breast carcinoma cell lines and one normal breast cell line, two lung carcinoma cell lines and one normal lung cell line, and two hepatoma cell lines and one normal liver cell line as shown below in detail: two human colon carcinoma cell line

(HT-29 & Colon 205) and one normal human colonic epithelial cell line (FHC); two human breast carcinoma cell lines (MDA-MB231 & ZR75) and one normal human mammary epithelial cell line (MCF-10A); two lung carcinoma cell lines (A549 & PE089) and one normal human lung cell line (HEL-229); two hepatoma cell lines (Hep3B & HepG2) and one normal human liver cell line (BNL cl.2). The following analyses are available to the above cell lines: 1. MTT assay:

The MTT assay is to test the capacity of each of four drugs to inhibit growth of cancer cells. After the cells are cultured in cell dishes, the drugs with different concentrations are added into the cell dishes for actions in following five days separately and then evaluations with the dye, that is, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT). As a yellow dye compound accepting hydrogen ions, MTT acting on respiratory chains in mitochondria of live cells is characteristic of tetrazolium split under actions of SDH (succinatedehydrogenase) as well as cytochrome C and production of blue formazan crystals which are in proportion to the count of live cells (note: MTT is not reduced by SDH which disappears in dead cells). Then, the count of live cells is evaluated in the absorbance test with formazan crystals dissolved by DMSO (BiBitill|il:ipli:dei/lf/The capacity of cells for reduction of MTT (that is, formazan crystals produced) is determined according to the O.D. value which means activity of mitochondria (count of live cells). Accordingly, the MTT assay is an indicator by which the growth of cells is determined.

In the assays of evaluating cancer cells killed by each of four anticancer drugs (Apiole, AP-02, AP-04 and AP-05), the four anticancer drugs with different concentrations such as 0, 0.1, 1, 10, 50 and 100 pg/ml are added into cancer cells and normal cells for actions in following 48 hours, respectively, a. Breast cell line:

As shown in FIG. 1A, the growth of breast carcinoma cells, MDA-MB-231, is not inhibited by Apiole. IC50 (half maximal inhibitory concentration) of other three drugs are 45.45 pg/ml (AP-02 with the optimal effect to inhibit growth of cancer cells), 373.40 pg/ml (AP-04) and 63.37 pg/ml (AP-05), respectively. Moreover, the growth of breast carcinoma cells, ZR75, is not inhibited by Apiole, AP-02, AP-04 or AP-05, as shown in FIG. IB. FIG. 1C presents experimental results that the growth of normal breast cells, MCF-10A, is not affected by Apiole. IC50 of other three drugs are 27.63 pg/ml (AP-02), 181.40 pg/ml (AP-04) and 26.44 pg/ml (AP-05), respectively. b. Lung cell line:

It can be seen from experimental results in FIG. 2A that the growth of lung carcinoma cells, A549, is not inhibited by Apiole. IC50 of other three drugs are 67.11 pg/ml (AP-02), 189.96 pg/ml (AP-04) and 80.93 pg/ml (AP-05), respectively. As shown in FIG. 2B, the growth of lung carcinoma cells, PE089, is not inhibited by Apiole or AP-04. IC50 of other two drugs are 38.36 pg/ml (AP-02 with the optimal effect to inhibit growth of cancer cells) and 50.11 pg/ml (AP-05), respectively. FIG. 2C presents experimental results that the growth of normal lung cells, HEL-299, is not affected by Apiole. IC50 of other three drugs are 10.20 pg/ml (AP-02), 37.14 pg/ml (AP-04) and 15.31 pg/ml (AP-05), respectively. c. Liver cell line:

As shown in experimental results in FIG. 3A, the growth of hepatoma cells, Hep3B, is not inhibited by Apiole or AP-04. IC50 of other two drugs are 22.61 pg/ml (AP-02 with the optimal effect to inhibit growth of cancer cells) and 53.79 pg/ml (AP-05), respectively. Moreover, the growth of hepatoma cells, HepG2, is not inhibited by Apiole, as shown in the another experimental result (FIG. 3B). IC50 of other three drugs are 40.21 pg/ml (AP-02), 81.04 pg/ml (AP-04) and 37.55 pg/ml (AP-05) , respectively.

It can be seen from the experimental results in FIG. 3C that the growth of normal liver cells, BNL cl.2, is not affected by Apiole or AP-04. IC50 of other two drugs are 67.74 pg/ml (AP-02) and 87.10 pg/ml (AP-05), respectively. d. Colon cell line:

As shown in FIG. 4A, the growth of colon carcinoma cells, Colon 205, is not inhibited by Apiole. IC50 of other three drugs are 3.31 pg/ml (AP-02 with the optimal effect to inhibit growth of cancer cells), 92.98 pg/ml (AP-04) and 22.30 pg/ml (AP-05), respectively. Moreover, the growth of colon carcinoma cells, HT-29, is not inhibited by Apiole, as shown in another experimental result (FIG 4B). IC50 of other three drugs are 7.69 pg/ml (AP-02 with the optimal effect to inhibit growth of cancer cells), 626.80 pg/ml (AP-04) and 11.27 pg/ml (AP-05), respectively.

It can be seen from experimental results in FIG. 4C that the growth of normal colon cells, FHC, is not affected by Apiole. IC50 of other three drugs are 52.77 pg/ml (AP-02), 101.30 pg/ml (AP-04) and 56.14 pg/ml (AP-05), respectively.

The above experimental results are summarized in Table 1 which presents AP-02 has the optimal effect of inhibiting growth of cells. Moreover, the growth of each of two colon carcinoma cells, Colon 205 and HT-29, inhibited is better than that of normal colon cells, FHC, inhibited. Accordingly, AP-02 and Colon 205 & HT-29 are selected as the drug and cell lines in experiments, respectively.

Table 1

Unit: pg/ml 2. Cell viability assay

The cell viability assay is to test the capacity of each of four drugs to kill off cancer cells. 8 x 105 cells selected from one of the two cell lines, Colon 205 and HT-29, which

were cultured in cell dishes, are distributed into seven 6-cm dishes. DMSO and AP-02 (lpg/ml (low dose), 5pg/ml (medium dose) and 30pg/ml (high dose), respectively) are dispensed to the dishes for cultures in following 48 hours. Then, dead cells and live cells, both of which are collected in the same tube, are counted with trypan blue. Having intact cell membranes for selective permeability, live cells are neither permeated by trypan blue, which is a macromolecular dye, nor stained blue; on the other hand, dead cells with ruptured cell membranes are permeated by trypan blue and stained blue. As such, the cell viability is determined.

As shown in FIG. 5, AP-02 performs well in killing off colon carcinoma cells such as Colon 205 and HT-29. The cell viabilities of colon carcinoma cells, Colon 205, in low-dose drugs, medium-dose drugs and high-dose drugs are 64.6%, 53.9% and 25.1%, respectively; the cell viabilities of colon carcinoma cells, HT-29, in low-dose drugs, medium-dose drugs and high-dose drugs are 89%, 66.9%. and 40.1%, respectively. 3. Cell cycle analysis & apoptosis analysis:

Based on experimental results for the MTT assay and the cell viability assay on cells, the cancer cell line most sensitive to medicines and the drug with the optimal effect to in-hibit/kill off cancer cells can be selected. With drugs (low-dose, medium-dose and high-dose, respectively) added into cancer cell lines, which were cultured in cell dishes, for actions in following 48 hours, the cancer cells are cut by trypsin, cleaned, immobilized with alcohol, and stained with pro-pidium iodide (PI). Because exciting light with the wave length of between 562 and 588 nm is released by PI inserted into DNA base-pairs and stimulated by 488 nm light, the cell cycle or the apoptosis of cancer cells under effect of drugs can be determined.

Based on experimental results for the MTT assay and the cell viability assay, Colon 205 is selected as the cell line for experiments and tested in FCM (flow cytometry) for the cell cycle and the apoptosis induced under effects of AP-02 (lpg/ml, 5pg/ml and 30pg/ml, respectively). 8 x 105 Colon 205 cells are distributed to eight 6-cm dishes; the serum starvation is enabled in starvation media in which 0.04 % FBS (fetal bovine serums) are included. After 16 hours, normal culture media (with 10 %FBS) are added into a dish of cells (so-called "Re-serum" cells)·, starvation media (with Θ. 0 4 %FBS) and DMSO are added into: another dish of cells, and AP-02 (Ipg/ml, Spg/ml and 30pg/ml) and Lope-01 (lpg/ml, Spg/ml and 30pg/ml) are added into other six dishes of cells, respectively. After cultivation in following 43 hours, the: cells are collected for the cell cycle analysis and the apoptosis: analysis in FGM.

It: can be seen from experimental results that the G1 arrest is observed at: cells in which starvation media are· added rather than at both the "Re-serum" cells: and any other cells: in which AP—02 with any distinct dose is added. Accordingly, the cell cycle of cancer cells on which AP-02 acted is unaffected, as shown in FIGI 6. On the other hand, the percentage of apoptosis: in "Sub-Gl" is raised when the doses of drugs are increased from low to high. After statistic analysis, the percentages of apoptosis in "Sub-Gl" are increased from 3% (AP-02;: lpg/ml) to 17.3% (AP-02; 30pg/ml).

Embodiment 2: Evaluation of the effect of drugs to inhibit tumors in vivo (experimentation on animals)

Based on the above experimental results, the cancer cell line most sensitive to medicines and the drug with: the optimal effect to: inhibit/kill off cancer cells are screened out for following experimentation on animals. As the experimental animal, each of 4-week-old nude mice is implanted with 2 x IO6 freshly cultured tdBd)ijjjjce:li((((sujsieh)Sid:ri:::::::at the dorsal part and grouped according to the measured size: of a grown tumor. There: are seven nude mice in each of four groups (2:8: mice totally) which are named as "vehicle", "high-dose drug", "medium-dose drug" and "low-dose drug", respectively. The administrations based on intraperitoneal ingestion are: available to the mice once a week but terminated until the size of a grown tumor under close and constant observation matches a desired size with which the efficiency of antibody inhibition is: evaluated.

As shown in experimental data in the MTT assay, IC50 of AP-02; dispensed is on the decrease from colon carcinoma cell lines to breast carcinoma cell lines, lung carcinoma cell lines and hepatoma cell lines. Accordingly, Colon 205 is selected as: the cancer cell line: for experimentation on animals: to which the administrations of AP-02 and Lope-01 based on intraperitoneal injection are available. In this regard, each of 4-week-old nude mice is implanted with 2 x 10e heterograft cells at the dorsal part and grouped according to the measured size of a grown tumor. The administrations based on intraperitoneal injection are available to seven nude mice in each of four groups (28 mice totally distributed to four groups named as "PBS (vehicle)", "AP-02 (1 mg/kg)", "AP-02 (5 mg/kg)" and "AP-02 (30 mg/kg)", respectively) twice a week and continued for six consecutive weeks after which the 28 mice are sacrificed. The tumor is estimated based on the following equation, tumor volume (mm3) = (w2 x 1)/2 where w and 1 are the width (mm) and the length (mm) of a tumor, respectively. Moreover, the weight of each mouse is checked and recorded during administrations for evaluation of drug toxicity.

As shown in experimental data in FIG. 7A, the growth rates of tumors on mice in "PBS" are faster than those of tumors on other mice fed with drugs and AP-02 (5 mg/kg) compared with other drugs performs better in inhibiting growth of tumors. It can be seen from data of mice (FIG. 7B) that the weights of mice on which tumors are grown in weeks decrease slightly and the weights of mice fed with AP-02 (5 mg/kg), which perform well in inhibiting growth of tumors compared with other drugs, decrease less than those of other mice. In summary, AP-02 (5 mg/kg) has the potential to serve as an effective clinical medication to treat colon carcinoma in the future.

Embodiment 3: Investigation of the tumor-suppressor mechanism of drugs based on experimentation on animals

In experimentation on animals, the tumors grown on two mice randomly selected from every group, "AP-02 (1 mg/kg)", "AP-02 (5 mg/kg)", "AP-02 (30 mg/kg)" and "PBS", are collected and ground with a homogenizer for extraction of proteins which are checked by the West blotting test that depends on apoptosis-related antibody to investigate the tumor-suppressor mechanism of drugs.

With GAPDH Glyceraldehyde-3-phosphate dehydrogenase/Bse-lected as the "internal control" among tumors on all groups of mice, the over expressions of apoptosis-relatedBprbOlhBlBidh a®p53, Bad, Bcl2 and Apaf 1 are detected in mice fed with AP02 (5 mg/kg) and the expressions of caspase 9, caspase 3 and caspase 8 in the active state are observed in mice fed with AP02 (1 mg/kg), as shown in experimental data in FIG. 8. Due to the over expression of tumor suppressor protein p53 in experimental results, AP02 is competent in inhibiting growth of tumors .

According to the above embodiments, AP-02 performs better in inhibiting growth of colon carcinoma cells such as Colon 205 and HT-29 rather than that of FHC. AP-02, which presents the best effect to kill off colon carcinoma cells, Colon 205, has no effect on the cell cycle of Colon 205. Moreover, inducing apoptosis of Colon 205, AP-02 (5 mg/kg) perform best in inhib iting growth of tumors but results in least weight loss of mice. Accordingly, the fact of growth of tumors inhibited by AP02 is demonstrated due to the over expression of tumor suppressor protein p53.

The conversion factor to convert weight from a mouse to a human being should be 12.3 according to "Estimating the maximum safe starting dose in initial clinical trials for therapeutics in adult healthy volunteers" published by the U.S. Food and Drug Administration in 2005. Accordingly, the effective dose administrated for a human being is between (0.2/12.3) and (120/12.3), i.e., between 0.016 and 9.56 (mg/kg) based on the effective dose of 0.2-120 mg/kg for a mouse; the better dose administrated for a human being is between (0.5/12.3) and (60/12.3), i.e., between 0.041 and 4.88 (mg/kg) based on the better dose of 0.5-60 mg/kg for a mouse; the optimal dose administrated for a human being is between (1/12.3) and (30/12.3), i.e., between 0.081 and 2.44 (mg/kg) based on the optimal dose 1-30 mg/kg for a mouse.

In summary, the Apiole derivatives with medical applications for preparation of anticancer drugs, which are innovative work in technical ideas and feature several effects in contrast to conventional anticancer drugs, meet novelty and non-obviousness for patentability.

Claims (12)

Use of apiol derivatives for the manufacture of a medicament for the treatment of anti-cancer drugs, the chemical structural formula being shown below: wherein R 1 and R 2 may be a hydrogen atom, an alkyl of one to three carbon atoms, an alkenyl of one to three carbon atoms, an alkynyl of one to three carbon atoms, or an ether of one to three carbon atoms. The use according to claim 1, wherein the apiol derivative is AP-02, which is 5-allyl-6-methoxybenzo [d] [1,3] dioxol, as represented in the following chemical structural formula: The use according to claim 1, wherein the apiol derivative is AP-04, which is 5-methoxybenzo [d] [1,3] dioxol, as represented in the following chemical structural formula: Use according to claim 1, wherein the apiol derivative is AP-05, which is 5-allylbenzo [d] [1,3] dioxol, as represented in the following chemical structural formula: The use according to claim 1, wherein the apiol derivative is each time characteristic of the effective dosage between 0.016 and 9.56 mg / kg. The use according to claim 1, wherein the apiol derivative is each time characteristic of the better dosage between 0.041 and 4.88 mg / kg. The use according to claim 1, wherein the apiol derivative is each time characteristic of the optimum dose between 0.081 and 2.44 mg / kg. The use of claim 1, wherein the apiol derivative is capable of inhibiting proliferation of hepatoma cells, colon cancer cells, lung cancer cells and breast cancer cells. The use according to claim 1, wherein the apiol derivative contributes to apoptosis to effect inhibition of tumor growth. The use according to claim 1, wherein the apiol derivative contributes to the overexpression of the tumor suppressor protein p53 to effect inhibition of tumor growth. The use according to claim 1, wherein the apiol derivative can be a pharmaceutically acceptable support agent, an excipient, or a diluent. The use according to claim 1, wherein the apiol derivative has the dosage form selected from a combination of a solution, a suspension liquid, an emulsion, powders, a pastille, a pill, syrups, a buccal tablet, a tablet, a chewable tablet, rigid pastes and a capsule.