Patent Application
20150366836
1. Technical Field
The present disclosure relates to a method and a pharmaceutical composition for treating colorectal cancer. In addition, the present invention relates to the establishment of animal model.
2. Description of Related Art
Colorectal cancer (including colon cancer and rectum cancer) is malignant tumor that comes from polypus in the colon. The mortality rate is the third place of all cancers in Taiwan when the patient is diagnosed with colorectal cancer in the later period or metastasis with low survival rate. Colorectal cancer proliferating to colon or rectal is characterized by cell accumulation near lymph nodes, and those cells accumulated near lymph nodes may invade and migrate through the lymphatic system to other organs or tissues such as liver.
As appreciated the difficulty in treating colorectal cancer, it is the first priority goal of the field to find novel and effective drugs. Furthermore, there is always a distinct gap between laboratory experiments and clinical use; therefore, it is also an important task to imitate the laboratory experiments as similar as possible with a realistic pathophysiological condition. In this consideration, animal model is indeed taken as a significant step before the candidate drugs actually enter clinical trials. Of course, the animal model itself has still certain level of difference from human body. However, the appropriate animal model can provide a suitable glance to mimic for the exploration of human body disease.
In light of the foregoing, there is always a need for novel and useful drug for treating colorectal cancer. In addition, it will be helpful for the drug screening if the conventional animal model can be modified to be more similar to human body.
One of the objects of the present invention is to provide a novel and useful drug for treating colorectal cancer by validating the medical use of 16-hydroxy-cleroda-3,13-dine-15,16-olide (HCD) in this regard.
Another object of the present invention is to provide a method for treating colorectal cancer, preferably the method can align the use of 16-hydroxy-cleroda-3,13-dine-15,16-olide with other conventional anti-cancer drugs to reduce the required dosage of said conventional anti-cancer drugs and obtain better efficacy.
More an object of the present invention is to establish an animal model having pathophysiological condition more similar to human.
In order to achieve the aforesaid objects, the present invention provides a pharmaceutical composition for colorectal cancer treatment, comprising: an effective amount of 16-hydroxy-cleroda-3,13-dine-15,16-olide; and a pharmaceutically acceptable carrier.
Preferably, said effective amount is 0.6 to 6.5 mg/kg body weight.
Preferably, said composition comprises 0.5 to 10 μM of said 16-hydroxy-cleroda-3,13-dine-15,16-olide.
Preferably, said pharmaceutically acceptable carrier is water, phosphate buffered saline, alcohol, glycerol, chitosan, alginate, chondroitin, Vitamin E, mineral oil, dimethyl sulfoxide (DMSO), or a combination thereof.
Preferably, an administration route of said composition is via oral administration, intravenous injection, intrathecal injection, intraperitoneal injection, or a combination thereof.
The present invention also provides a method for treating a colorectal cancer, comprising: administrating an object in need an effective amount of 16-hydroxy-cleroda-3,13-dine-15,16-olide.
Preferably, said effective amount is 0.6 to 6.5 mg/kg body weight.
Preferably, said administrating is via oral administration, intravenous injection, intrathecal injection, intraperitoneal injection, or a combination thereof.
Preferably, said 16-hydroxy-cleroda-3,13-dine-15,16-olide is administrated with a pharmaceutically acceptable carrier
Preferably, said pharmaceutically acceptable carrier is water, phosphate buffered saline, alcohol, glycerol, chitosan, alginate, chondroitin, Vitamin E, mineral oil, dimethyl sulfoxide (DMSO), or a combination thereof.
Preferably, said method further comprises a step after administrating said 16-hydroxy-cleroda-3,13-dine-15,16-olide: administrating said object with 5-Fluorouracil.
The present invention more provides a method for establishing an animal model bearing an enteritis: (a) providing an animal; (b) administrating said animal with azoxymethane; and (c) administrating said animal with a dextran sodium sulfate solution.
Preferably, said administrating in step (b) is an intraperitoneal injection.
Preferably, a dosage of said azoxymethane in step (b) is 8 to 12 mg/kg body weight.
Preferably, said administrating in step (c) is oral administration via drinking water.
Preferably, said dextran sodium sulfate solution in step (c) has a concentration of 1 to 3 wt %.
Preferably, said step (c) is repeated at least once, and said method further comprises a resting period between repeats of said step (c); wherein said resting period is referred to as a period that said animal is not administrated with said azoxymethane and said dextran sodium sulfate solution.
Preferably, said enteritis is inflammatory bowel disease.
Preferably, said animal is rabbit, pig or rodent.
To sum up, the present invention validates the medical use of HCD in treating colorectal cancer and its ability to reduce required dosage of conventional anti-cancer drugs. Moreover, the present invention also establishes an animal model bearing enteritis and being patho-physiologically more similar to human than conventional animal models so that the drug screening using said animal model can be more effective and reliable in subsequent clinical trials.
16-hydroxy-cleroda-3,13-dine-15,16-olide (HCD) isolated from Polyalthia longifolia possess some medicinal values; however, there is no evidence showing its value in treating colorectal cancer before the present invention. The term “colorectal cancer” herein is referred to as colon cancer, rectum cancer, or a combination thereof.
The term “treatment or treating” herein is referred to control or reduce the size of the tumor, prevent or limit the metastasis of the cancer cells, or a combination thereof. The term “effective amount” herein is referred to as an amount of the active ingredient that is sufficient to perform the aforesaid efficacies of treatment.
Said effective amount can be obtained from clinical trial, animal model, or in vitro cell culture data. It is known in the field that the effective amount obtained from animal model or in vitro cell culture data can be calculated into the effective amount suitable for human use. For instance, as reported by Reagan-Shaw et al., 2008, “μg/ml” (effective amount based on in vitro cell culture experiments)=“mg/kg body weight/day” (effective amount for mouse). Furthermore, the effective amount for mouse can be further modified based on the fact that the metabolism rate of mice is 6 times fast compared to human.
Said pharmaceutically acceptable carrier in the present invention includes but not limited to water, phosphate buffered saline, alcohol, glycerol, chitosan, alginate, chondroitin, Vitamin E, mineral oil, dimethyl sulfoxide (DMSO), or a combination thereof. Generally, the pharmaceutically acceptable carrier can be chosen based on the desired administration route, components of the drug, treatment strategies, or purposes to be met.
The first aspect of the present invention is to provide a pharmaceutical composition for colorectal cancer treatment. Said pharmaceutical composition comprises 16-hydroxy-cleroda-3,13-dine-15,16-olide (HCD) as the active ingredient. The effective amount of said HCD is 0.6 to 6.5 mg/kg body weight. The pharmaceutical composition can be administrated via oral administration, intravenous injection, intrathecal injection, intraperitoneal injection, or a combination thereof.
The second aspect of the present invention is to provide a method for treating a colorectal cancer, comprising: administrating an object in need an effective amount of 16-hydroxy-cleroda-3,13-dine-15,16-olide. The effective amount of said HCD is 0.6 to 6.5 mg/kg body weight. The administrating can be via oral administration, intravenous injection, intrathecal injection, intraperitoneal injection, or a combination thereof.
In a preferable embodiment of the present invention, a treating strategy is provided. Said treating strategy is to reduce the required dosage of a conventional anti-cancer drug. Said treating strategy comprises a pre-treatment and a subsequent treatment. Said pre-treatment is administrating an object in need an effective amount of 16-hydroxy-cleroda-3,13-dine-15,16-olide. Said subsequent treatment is administrating said object with a conventional anti-cancer drug. After said pre-treatment, the data of the present invention showed that the efficacy of said anti-cancer drug can be improved and the dosage required for the efficacy can be reduced.
Taking 5-Fluorouracil (5-FU, which is a known drug for chemotherapy for colorectal cancer) as an example, the present invention showed (in the following data) pre-treating with HCD can enhance the efficacy of the subsequent 5-FU treatment. The data further showed that the pre-treatment of Prodigiosin (PG, as a positive) can also provide similar effects in enhancing the efficacy of the subsequent 5-FU treatment. Accordingly, the present invention indicates the potential of a co-treating strategy having a pre-treatment of HCD or PG and a subsequent treatment of an anti-cancer drug.
The third aspect of the present invention is to establish an animal model bearing enteritis. In a preferable embodiment of the present invention, said animal is not immunodeficient, which means said animal is normal in immunological competence. In this way, variances interfering with the experiments can be reduced and the drug screening data from said animal model can be more likely to be the things happened in human body. In an alternative embodiment, said animal can be rabbit, pig or rodent.
In a preferable embodiment, after said animal is ready, the present method for establishing said animal model can be separated into three periods of induction:
The first period is to administrate said animal with azoxymethane (AOM). Said azoxymethane can be administrated by intraperitoneal injection. Preferably, a dosage of said azoxymethane is 8 to 12 mg/kg body weight.
The second period is to administrate said animal with a dextran sodium sulfate solution (DSS solution). Preferably, said dextran sodium sulfate solution is taken as the daily drinking water of said animal, which means said dextran sodium sulfate solution is administrated by oral administration. Preferably, said dextran sodium sulfate solution in step (c) has a concentration of 1 to 3 wt %.
The third period is a resting period. Said resting period is referred to as a period that said animal is not administrated with said azoxymethane and said dextran sodium sulfate solution.
In a preferable embodiment of the present invention, the second period is repeated at least once and said resting period is conducted between repeats of said second period. Said “conducted between repeats” is referred to that the resting period can be conducted between every repeat of said second period or between preceding repeats and subsequent repeats.
For instance, said second period of administrating said animal with a dextran sodium sulfate solution is conducted once a day for 7 successive days, which is recognized as said second period is repeated 7 times. Then, said third period (resting period) is conducted to “rest” said animal for 7 successive days. After that, another said second period was conducted for additional 7 successive days.
The 16-hydroxy-cleroda-3,13-dine-15,16-olide (HCD) used in the present study was obtained from Professor Yi-Chen Chia (Department of Food Science & Technology, Tajen University, Taiwan). The Prodigiosin (PG) isolated from Serratia marcescens was obtained from Professor Jui-Hsin Su (Institute of Marine Biotechnology, National Drug Hwa University, Taiwan). The known drug for chemotherapy, 5-Fluorouracil (5-FU) was purchased from Sigma. Said HCD, PG, and 5-FU of various concentrations were dissolved in phosphate buffered saline (PBS) as indicated in the following paragraphs and were sterilized before use.
Two cell lines were used in this study, Caco2 cells and HT-29 cells. Both of them are colorectal cancer cells. Caco2 cells and HT-29 cells were maintained in DMEM (or RPMI) supplemented with 20% or 10% fetal bovine serum (FBS; GIBCO), pH 7.4 at 37° C. with continuous circulation of 5% CO2 incubator. The medium was changed every 2 days and the cells were trypsinized using trypsin/EDTA when reaching 80%-90% confluence.
Caco2 cells and HT-29 cells were treated by the following treatment (Table 1) for 24, 36 or 48 hr for examining the effects of HCD, PG, and Dox on their viability.
FU1 is referred as FU of 1 μM; FU2 is referred as FU of 2 μM; FU10 is referred as FU of 10 μM; FU50 is referred as FU of 50 μM; FU100 is referred as FU of 100 μM.
HCD0.5 is referred as HCD of 0.5 μM; HCD1 is referred as HCD of 1 μM; HCD2 is referred as HCD of 2 μM; HCD5 is referred as HCD of 5 μM; HCD10 is referred as HCD of 10 μM.
PG0.5 is referred as PG of 0.5 μM; PG1 is referred as PG of 1 μM; PG2 is referred as PG of 2 μM; PG5 is referred as PG of 5 μM; PG10 is referred as PG of 10 μM.
In this example, the effects of HCD, PG, and 5-FU on the viability of Caco2 cells and HT-29 cells were examined. The MTT assay was employed for this purpose. The MTT (3-(4-,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay is a common colorimetric method in the field for cell viability analysis. MTT (yellow tetrazolium salt) is reduced to a purple formazan by living cells and detection to the purple formazan can be calculated as the cell viability.
Briefly, cells (Caco2 or HT-29) were seeded in 96-well plate (7×103 cell per well) and incubated overnight (37° C., 5% CO2). Then, cells were treated for 24, 36, or 48 hr in accordance with the treatments listed in the Table 1. The results were showed in
The IC50 of 5-FU was 100 μM and 50 μM in Caco2 cells (48 hr treatment) and HT-29 cells (36 hr treatment) respectively. The IC50 of PG was 2 μM and 0.5 μM in Caco2 cells (48 hr treatment) and HT-29 cells (36 hr treatment) respectively. The IC50 of HCD was 5 μM in Caco2 cells (48 hr treatment) and HT-29 cells (36 hr treatment) respectively. The results in
On the other hand, in the pretreatment analysis, cells (Caco2 or HT-29) were seeded in 96-well plate (7×103 cell per well) and incubated overnight (37° C., 5% CO2). In according to the treatments listed in the Table 1, Cells were pre-treated by HCD or PG for 6, 12, 24, 36, or 48 hr and then were treated by 5-FU for additional 24 hr. The results were showed in
The results in
In light of the foregoing, HCD showed reliable effects on the viability of colorectal cancer cells. Furthermore, our data indicated a pre-treatment strategy with a pre-treatment of HCD or PG before 5-FU treatment provided improved effects on colorectal cancer cells viability.
The results of Example 2 in cell viability hinted the effects of HCD, PG, and 5-FU on arresting cell cycle. In this example, the phase of cell cycle of Caco2 and HT-29 cells after the treatment listed in Table 1 was determined by flow cytometer. Briefly, 7×104 cells per well were inoculated in 12-wells plate and incubated overnight at 37° C., 5% CO2. Then, cells were treated according to the treatment listed in Table 1 by indicated time period. After treatment, cells were harvested by trypsin and fixed with 70% ethanol at −20° C. for at least 3 hr. The cells were washed in cold PBS twice and then incubated with 1 mL (v/v) staining solution (20 μg/mL propidium iodide (PI), 0.1% Triton X-100, 0.2 mg/mL Rnase) at 37° C. for 30 minutes. Lastly, cells were analyzed by flow cytometer (Cytomics™ FC500, Backman, Fullerton, Calif., USA). Data from 10,000 cells were collected for each experimental group.
The following Tables 2 to 3 showed that the treatment of 5-FU increased cell cycle arrest at sub-G1 phase in both of the Caco2 and HT-29 cells. Tables 4 to 7 showed that, in both of the Caco2 and HT-29 cells, both of the treatment of HCD and PG increased cell cycle arrest at sub-G1 phase comparing with the control group in a dose- and time-dependent manner.
Please also refer to the following Tables 8-11. In terms of the pre-treatment strategy, it was noted that G2/M phase in Caco2 cells was decreased with 12 and 24 hr pretreatment of 1 or 2 μM of HCD and PG. While in HT-29 cells, pre-treatment of HCD or PG showed increase of sub G1 phase and decrease of G2/M phase. The results were consistent with the data obtained in Example 2, showing that the pre-treatment strategy is applicable for enhancing the effects of known drug, 5-FU.
16 ± 3.99
16 ± 3.99
A mouse model bearing inflammatory bowel disease (IBD) is a common and reliable animal model for in vivo cancer research. However, the conventional mouse model bearing IBD is not ideal because it requires several months to establish the mouse model. In addition, another concern of the conventional mouse model is that those mouse models are established in mice with immunodeficiency, which means the physiological condition of the experimental animals are not “normal” and therefore have more variance than expecting. In this example, a mouse model was established in a “normal mouse” by a more time-efficiency manner.
The animal experiments were approved by the National Dong-Hwa University Animal Ethics Committee and the experimental protocols were used according to the “Guide for the Care and Use of Laboratory Animals” of National Dong-Hwa University. On day 0, C57/BL6 mice (6-8 weeks old) were weighted and injected (intraperitoneal; i.p.) with 10 mg/kg body weight of azoxymethane (AOM) and then fed with 2% Dextran sodium sulfate solution (DSS solution) via water every day for 7 days. On day 8, the 2% DSS solution was changed to normal water for another 7 days. The cycle of DSS solution and water was repeated and the induction procedure was completed on day 35.
During the induction procedure, the body weight of the experimental mice was recorded and compared with mice of control group. The results were showed in
On day 35, the mice were sacrificed. The colon, spleen, liver, and kidney thereof were immediately removed, observed, and further examined by H&E staining.
The aforesaid data showed that the induction procedure was success in establishing IBD in the experimental mice.
After the establishment of IBD in the experimental mice (on day 35), the mice were injected (i.p.) with 5-FU (15 mg/kg body weight) or HCD (0.64, 1.6, or 6.4 mg/kg body weight) once every three days for additional 30 days. Then, the mice were sacrificed. The colon, spleen, liver, and kidney thereof were immediately removed, observed, and further examined by H&E staining.
During the experiment period, the body weight of the mice was recorded. The result in
The H&E staining of the intestine vertical section of the mice were showed in
