Patent Application
20170105999
The present invention relates to a method for treating a drug resistant cancer with an anti-emetic drug in combination of a chemotherapeutic drug.
Chemotherapy, particularly with a combination of anti-cancer agents, is the treatment of choice for delocalized tumors that are untreatable by surgery or radiation. However, some patients relapse after even a short period of time, and do not respond to a second course of chemotherapy.
Most malignant tumors show some sensitivity to cytotoxic drugs. Accordingly, treatment with these drugs generally provides remission and shrinkage of the tumor that may last for weeks to months. Nonetheless, in many cases the tumors regrow, and this regrowth is resistant to further cytotoxic treatments.
Theoretically it should be possible to solve this problem by administering a combination of drugs that act differently. This method of treatment is based upon the extremely small probability that two or more different drug resistances would arise spontaneously in the same cell. Combination chemotherapy appeared to obviate the problem of drug-resistant tumor cells.
Research was then directed to protocols for administering anticancer drugs in combinations. Newly developed drugs and combination chemotherapy several decades ago produced high cure rates for some childhood leukemia and for Hodgkin's disease. However, the major killers, such as lung cancer, breast cancer, and cancers of the gastrointestinal tract, remained resistant to chemotherapy. The failures of combination chemotherapy were not understandable. Many theories were proposed to explain the observations, but few of these theories could be adequately tested.
Although significant strides have been made in the development of pharmaceutical compositions for treating cancer, new methods of treating drug resistant cancer are required.
According to the invention, it was unexpectedly found that prochlorperazine in combination of a chemotherapeutic drug exhibits a synergistic effect in reducing the size and number of the cancer cells, and inhibiting the growth of cancer cells with drug resistance properties.
In one aspect, the invention provides a method for treating a subject with a cancer resistant to a chemotherapeutic drug. The method comprises administering to said subject a therapeutically effective amount of prochlorperazine or its analog or metabolite, or a pharmaceutically acceptable salt thereof, in combination of the chemotherapeutic drug.
In one further aspect, the invention provides a method for preventing cancer metastasis. The method comprises administering to a subject in need thereof a therapeutically effective amount of prochlorperazine or its analog or metabolite, or a pharmaceutically acceptable salt thereof, in combination of a chemotherapeutic drug
In one more aspect, the invention provides a pharmaceutical composition or combination for treating a subject with a cancer resistant to a chemotherapeutic drug or for preventing cancer metastasisc, comprising a therapeutically effective amount of prochlorperazine or its analog or metabolite, or a pharmaceutically acceptable salt thereof, in combination of a chemotherapeutic drug, in combination of a chemotherapeutic drug.
In one yet aspect, the invention provides a use of prochlorperazine or its analog or metabolite, or a pharmaceutically acceptable salt thereof, for manufacturing a medicament for treating a subject with a cancer resistant to a chemotherapeutic drug in combination of a chemotherapeutic drug.
In one further yet aspect, the invention provides a use of prochlorperazine or its analog or metabolite, or a pharmaceutically acceptable salt thereof, for manufacturing a medicament for preventing cancer metastasis in combination of a chemotherapeutic drug.
In one embodiment of the invention, the chemotherapeutic drug is selected from the group consisting of gefitinib, erlotinib, afatinib, pemetrexed, cisplatin, paclitaxel, docetaxel, gemcitabine, navelbine, irinotecan, avastin, 5-fluorouracil, methotrexate, oxaliplatin, tegafur-gimeracil-oteracil potassium (TS-1), and epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors and combination thereof.
The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended drawing. In the drawings:
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which this invention belongs.
As used herein, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a sample” includes a plurality of such samples and equivalents thereof known to those skilled in the art.
As used herein, the term “prochlorperazine” refers to a dopamine (D2) receptor antagonist that is used for the antiemetic treatment of nausea and vertigo. Prochlorperazine has the structure of
As used herein the term “pharmaceutically acceptable salt” refers to any pharmaceutically acceptable salt of prochlorperazine. The pharmaceutically acceptable salts include ammonium salts, alkali metal salts such as potassium and sodium (including mono, di-and tri-sodium) salts (which are preferred), alkaline earth metal salts such as calcium and magnesium salts, salts with organic bases such as dicyclohexylamine salts, N-methyl-D-glucamine, and salts with amino acids such as arginine, lysine, and so forth.
As used herein, the term “metabolite” refers to any intermediate and product of metabolism. Some examples of the metabolites of prochlorperazine include but are not limited to N-desmethyl prochlorperazine, prochlorperazine sulfoxide and prochlorperazine sulfoxide 4′-N-oxide. In one particular example, the metabolite is N-desmethyl prochlorperazine.
As used herein, the term “analog” refers to any compound with an altered chemical structure having the same function or activity. Some examples of the analog of prochlorperazine include but are not limited to the compounds having the following structures:
As used herein, the term “subject” refers to any warm-blooded species such as humans and animals. The subject, such as a human, to be treated according to the present invention may in fact be any subject of the human population, male or female, which may be divided into children, adults, or elderly. Any one of these patient groups relates to an embodiment of the invention.
The invention provides a method for treating a subject with a cancer resistant to a chemotherapeutic drug. The method comprises administering to said subject a therapeutically effective amount of prochlorperazine or its analog or metabolite, or a pharmaceutically acceptable salt thereof, in combination of the chemotherapeutic drug.
In some examples of the present invention, the combination of prochlorperazine and a chemotherapeutic drug exhibits a synergistic effect in reducing the size and number of the cancer cells. In other examples of the present invention, the combination of prochlorperazine and a chemotherapeutic drug exhibits a synergistic effect in inhibiting the growth of cancer cells.
As used herein, the term “chemotherapeutic drug” refers to any drug providing anti-cancer effect, including but not limited to gefitinib, erlotinib, afatinib, pemetrexed, cisplatin, paclitaxel, docetaxel, gemcitabine, navelbine, irinotecan, avastin, 5-fluorouracil, methotrexate, oxaliplatin, tegafur-gimeracil-oteracil potassium (TS-1), and epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors and combination thereof. Preferred examples include gefitinib, erlotinib, afatinib, pemetrexed, cisplatin, 5-fluorouracil, irinotecan, paclitaxel, gemcitabine, avastin, TS-1, oxaliplatin. In one particular example, the chemotherapeutic drug is gefitinib.
It was found in the examples of the invention that some metabolites have the same cytotoxic activities as prochlorperazine, as shown in Table 2.
It was also found in the examples of the invention that some analogs have the same clonogenic activities as prochlorperazine, as shown in Table 3.
As used herein, the term “therapeutically effective amount” refers to an amount sufficient for providing an effect in treatment for a cancer, which is depending on the mode of administration and the condition to be treated, including age, body weight, symptom, therapeutic effect, administration route and treatment time.
In the present invention, various cancers can be treated with the method according to the invention. In some embodiments, the cancer is a solid cancer such as a solid tumor. In other embodiments, the cancer is “liquid” cancer or a hematological cancer. The cancer is selected from the group consisting of lung cancer, liver cancer, colorectal cancer, brain cancer, breast cancer, pancreatic cancer, gastric cancer, adenocarcinoma, squamous cell carcinoma, and large cell carcinoma.
It is found in the example of the invention that prochlorperazine exhibited cytotoxicity in various types of cancers, including adenocarcinoma, squamous cell carcinoma, large cell carcinoma, liver cancer, colorectal adenocarcinoma, brain cancer, breast cancer, pancreatic cancer, and myeloma, see Table 1.
The present invention provides a method treating a subject with a cancer resistant to a chemotherapeutic drug. The method comprises administering to said subject a therapeutically effective amount of prochlorperazine or its analog or metabolite, or a pharmaceutically acceptable salt thereof, in combination of the chemotherapeutic drug. In one example of the invention, the caner is a lung cancer such as non-small cell lung carcinoma (NSCLC).
On the other hand, the present invention provides a method for preventing cancer metastasis. The method comprises administering to a subject in need thereof a therapeutically effective amount of prochlorperazine or its analog or metabolite, or a pharmaceutically acceptable salt thereof, in combination of a chemotherapeutic drug. In one example of the invention, the caner is a lung cancer such as lung squamous cell carcinoma. In another example of the invention, the cancer is gastric cancer, such as signet ring cell carcinoma.
In one example of the invention, prochlorperazine in combination of erlotinib provided an effect in prevention of metastasis of a lung cancer, such as lung squamous cell carcinoma. In another example of the invention, prochlorperazine in combination of a chemotherapeutic drug, which is selected from the group consisting of cisplatin, 5-fluorouracil, irinotecan, paclitaxel, gemcitabine, avastin, TS-1, oxaliplatin and combination thereof, provided an effect in prevention of metastasis of a gastric cancer, such as signet ring cell carcinoma.
In addition, the invention provides a use of prochlorperazine or its analog or metabolite, or a pharmaceutically acceptable salt thereof for manufacturing a medicament for treating a subject with a cancer resistant to a chemotherapeutic drug. The invention also provides a use of prochlorperazine or its analog or metabolite, or a pharmaceutically acceptable salt thereof for manufacturing a medicament for preventing cancer metastasis.
In the invention, prochlorperazine or its analog or metabolite, or a pharmaceutically acceptable salt thereof (“the active compound”) may be formulated in a pharmaceutical composition or formulation, which may be administered in any route that is appropriate, including but not limited to oral or parenteral administration. In one example of the invention, the composition or formulation comprising prochlorperazine or its analog or metabolite, or a pharmaceutically acceptable salt thereof is administered through oral route, which may he in a solid or liquid form. The solid compositions or formulations include tablets, pills, capsules, dispersible powders, granules, and the like. The oral compositions also include gargles which are to be stuck to oral cavity and sublingual tablets. The capsules include hard capsules and soft capsules. In such solid compositions or formulations for oral use, one or more of the active compound(s) may be admixed solely or with diluents, binders, disintegrators, lubricants, stabilizers, solubilizers, and then formulated into a preparation in a conventional manner. When necessary, such preparations may be coated with a coating agent, or they may be coated with two or more coating layers. On the other hand, the liquid compositions for oral administration include pharmaceutically acceptable aqueous solutions, suspensions, emulsions, syrups, elixirs, and the like. In such compositions, one or more of the active compound(s) may be dissolved, suspended or emulsified in a commonly used diluent (such as purified water, ethanol or a mixture thereof, etc.). Besides such diluents, said compositions may also contain wetting agents, suspending agents, emulsifiers, sweetening agents, flavoring agents, perfumes, preservatives and buffers and the like.
The pharmaceutical compositions for parenteral administration include solutions, suspensions, emulsions, and solid injectable compositions that are dissolved or suspended in a solvent immediately before use. The injections may be prepared by dissolving, suspending or emulsifying one or more of the active ingredients in a diluent. Examples of said diluents are distilled water for injection, physiological saline, vegetable oil, alcohol, and a combination thereof. Further, the injections may contain stabilizers, solubilizers, suspending agents, emulsifiers, soothing agents, buffers, preservatives, etc. The injections, are sterilized in the final formulation step or prepared by sterile procedure. The pharmaceutical composition of the invention may also he formulated into a sterile solid preparation, for example, by freeze-drying, and may be used after sterilized or dissolved in sterile injectable water or other sterile diluent(s) immediately before use.
The examples below are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. Without further elaboration, it is believed that one skilled in the art can, based on the description herein, utilize the present invention to its fullest extent.
I. Cell Culture and Chemicals
A549, CL141 and H441 are EGER-wild type adenocarcinoma cell lines, HCC827 has EGFR-exon 19 deletion, and CL97 is an EGFR T790M and G719A mutations cell line. CL152, H2170 and H226 are squamous cell carcinoma cell lines and H1299 is a non-small cell lung carcinoma cell line. A549-ON cell line is A549 cell overexpression Oct4 and Nanog which we regarded it as cancer stem cell-like cell line (22). All cell lines were maintained in RPMI medium and supplemented with 10% fetal bovine serum (FBS, Invitrogen), 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin. For cell culture experiment, 10 mM prochlorperazine stock solution, was dissolved in dimethyl sulfoxide (DMSO; Sigma).Prochlorperazine, Cisplatin, Gemcitabine were purchased from Sigma.
II. Cytotoxicity and Sulforhodamine B Assay
Cells were plated in 96-well plates at a density of 2000 cells per well in triplicate. The cells were treated on the third day (to ensure proper plating efficiency and vitality) to indicated agents for 48 hrs. Cells were treated with different concentrations of prochlorperazine, cisplatin, gemcitabine, or a combination of, for example, prochlorperazine and gemcitabine. Cytotoxicity was assessed using the sulforhodamine B (SRB) assay (23). Briefly, the medium was discarded, and the adherent cells were fixed by 100 μl of cold 10% trichioroacetic acid (w/v) in each well for 1 h at 4° C. After fixation, cells were stained with 100 μl/well of 0.4% (w/v, in 1% acetic acid) SRB solution for 30 min at room temperature, and then washed 5 times with 1% acetic acid. After air-drying, 100 μl of 10 mM Tris base was added to each well and the absorbance was read at 530 nm. Cytotoxicity is expressed as the percent of cells in drug treated wells relative to number of cells in the solvent only control (set to 100%). Each experiment was performed independently at least 2 times in triplicate and cytotoxicity is given as means ±SD.
III. Clonogenic Assay
Tested cells were seeded respectively in 6 well plates with 104 cells per well for 14 days. Prochlorperazine and other tested drugs were added 24 hours after seeding of the cells. The medium and prochlorperazine were changed every 4 days. After the treatments, cells were washed with PBS, and the colonies were fixed with fix solution (methanol: acetic acid=3:1) and stained with 0.5% crystal violet in methanol. After removing the crystal violet carefully and rinse with tap water, the colonies were counted manually. Each experiment was performed independently at least 2 times in triplicate and cytotoxicities are given as means ±SD.
IV. Tumor Spheroid Assay
In brief, single cells were plated in 6-well ultralow attachment plates (Coming Inc.) at a density of 2,000 cells/mL in tumor spheroid culture medium, DMEM/F12 supplemented with 1% N2 Supplement (Invitrogen), 10 ng/mL basic fibroblast growth factor (Sigma-Aldrich), 10 ng/mL epidermal growth factor (Invitrogen) with 1% penicillin/streptomycin (Invitrogen) at 37° C. in a humidified atmosphere of 95% air and 5% CO2. Cells were cultured twice per week. When passaged, tumor spheres were harvested. Spheroids were dissociated with TrypLE™ (Invitrogen). Spheroids cell counting using the Trypan Blue Exclusion method.
V. Side Population Analysis and Purification Using Flow Cytometry
Single-cell suspensions of cells were detached from dishes with Trypsin-EDTA (Invitrogen) and suspended at 1×106 cells/mL in Hank's balanced salt solution (HIM) supplemented with 3% fetal calf serum and 10 mM Hepes. These cells were then incubated at 37° C. for 90 minutes with 20 μg/mL Hoechst 33342 (Sigma Chemical, St. Louis, Mo.). The ABC transporter inhibitor verapamil (Sigma) was added at a final concentration of 50 μM to confirm the gating area on flow cytometry. After 90 minutes incubation with indicated drugs, the cells were centrifuged immediately for 5 minutes at 300 g and 4° C. and resuspended in ice-cold HBSS, The cells were kept on ice to inhibit efflux of the Hoechst dye, and 1 μg/mL propidium iodide (PI, BD) were added to discriminate dead cells. Finally, these cells were filtered through a 40 μm cell strainer (BD) to obtain single-suspension cells. Cell dual-wavelength analysis and purification were performed on a dual-laser FACS Vantage SE (BD). Hoechst 33342 can be excited at 355 nm UV light and emitted blue fluorescence with a 450/20 band-pass (BP) filter and red fluorescence with a 675 nm edge filter long-pass (EFLP). A 610 nm dichroic mirror short-pass (DMSP) was used to separate the emission wavelengths. PI-positive (dead) cells were excluded from the analysis.
VI. Aldefluor Assay
High aldehyde dehydrogenase (ALDH) enzyme activity was used to detect ung cancer stem cell populations. The Aldefluor assay was performed according to the manufacturer's guidelines (StemCell Technologies). Briefly, single cells obtained from cell cultures were incubated in an Aldefluor assay buffer containing an ALDH substrate (bodipy-aminoacetaldehyde, BAAA) for 50 minutes at 37° C. As a negative control, a fraction of cells from each sample was incubated under identical conditions in the presence of an ALDH inhibitor (diethylaminobenzaldehyde, DEAB). Flow cytometry was used to measure the ALDH-positive cell population.
VII. In Vivo Examination of Anti-Lung Cancer Effects Mediated by Prochlorperazine.
In the first trial, human lung cancer cell line NCI-H441 (purchased from ATCC, 1 million cells/injection) cells were subcutaneously injected into the right flank of NOD/SCID mice (female, 4-6 weeks old). When tumors became palpable, their sizes were recorded using caliper and mice were randomly divided into control group (DMSO vehicle) and prochlorperazine treatment group (5 mg/kg, 5 days/week, i.p injection). Over the period of 4 weeks, tumorigenesis in both groups was measured using a caliper on a weekly basis. The change in tumor size was expressed as in fold change and plotted over time. Prochlorperazine treatment appeared to suppress and/or delay the growth of tumor as compared to the vehicle control (**p<0.01).
In the first drug combination test, NCI-H441 cells expressing firefly luciferase (6×105 cells/injection) were injected into NOD/SCID mice (4-6 week of age) via the lateral tail vein for tumor establishment. One week post tumor injection, mice were randomly divided into different groups: vehicle, prochlorperazine (1 mg/kg) in combination with pemetrexed (1 mg/kg) (a standard chemotherapeutic agent for NSCLC), pemetrexed (1 mg/kg)+cisplatin (1 mg/kg) and pemetrexed (1 mg/kg)+prochlorperazine (1 mg/kg) treatment groups. Tumor burden from different groups were recorded by caliper. The change in tumor size was expressed as in fold change and plotted over time. Subsequently, we performed another set of drug combination test. We used two major standard chemotherapeutic regimens in combination with prochlorperazine. For lung adenocarcinoma, the standard drug combination is pemetrexed and cisplatin whereas are used gemcitabine and cisplatin for lung squamous carcinoma. In CL97 (adenocarcinoma) tumor-bearing mice, the combination of pemetrexed+cisplatin+prochlorperazine provided the most significant tumor suppressive effect than pemetrexed+cisplatin and prochlorperazine alone groups. In CL152 (squamous) tumor-bearing mice, again standard regimen (gemcitabine+cisplatin) added with prochlorperazine appeared to suppress tumorigenesis better than prochlorperazine alone and gemcitabine+cisplatin groups. Next, we demonstrated that prochlorperazine sensitized gefitinib-resistant CL97 lung cancer cells in vivo. CL97 cancer cells (1.5×106 cells per injection) were injected subcutaneously into NOD/SCID mice to establish in viva tumor model. Mice were randomly divided into 4 groups: Control, gefitinib alone (100 mg/kg, PO, 5 times/week), prochlorperazine alone (5 mg/kg, IP, 5 times/week) and gefitinib+prochlorperazine groups. Significant tumor suppressive effect exerted by prochlorperazine alone and gefitinib +prochlorperazine groups were observed 5-week post tumor injection. By week 6 post tumor injection, gefitinib+prochlorperazine group showed the most significant tumor suppressive effect followed by prochlorperazine alone group while both control and gefitinib alone groups demonstrated a similar tumor burden.
Results
Cytotoxicity of Prochlorperazine in NSCLC Cell Lines
We first investigated the cytotoxic effect of prochlorperazine in various NSCLC cell lines. A549, A549-ON, CL97, CL141, HCC827, and H441 were lung adenocarcinoma cell lines. CL152, H2170, and H226 were squamous cell carcinoma cell lines. Cell viability was determined by SRB assay. After treatment for 48 hours with prochlorperazine with various concentrations, the IC50 values in all cancer cell lines were around 20 μM (see Table 1). The experiments were perfoinied to determine the anti-tumor activities of prochlorperazine using clonogenic assay. Clonogenicity of NSCLC cell lines was reduced in a concentration-dependent manner after exposure to different concentration of prochlorperazine Cancer stem cell (CSC) postulates the existence of a tumor cell population uniquely endowed with self-renewal capacity and therapy resistance. Since prochlorperazine was predicted to preferentially inhibit the lung CSC, we next examined whether the proportion of NSCLC (a lung squamous cell carcinoma cell with wild-type EGFR) spheres could be diminished by prochlorperazine treatment. To determine whether side population (SP) cells, which represent a cell population with CSC characteristics, exist in NSCLC cell lines, we stained the cells with the fluorescent dye, Hoechst 33342, and analyzed the cells with flow cytometry. After the exclusion of dead cells and cellular debris based on the scatter signals, the NSCLC cells contained a small population of cells with SP cell characteristics. After 48 hours of incubation with prochlorperazine at 2.5, 5 and 10 μM, the proportion of SP cells were dose-dependently decease (see Table 1).
Prochlorperazine reduces the proportion of side population cells and ALDH+ cells
It was also investigated whether prochlorperazine treatment could deplete the percentage of the cells with ALDH expression (ALDH is an established marker for both hematopoietic and NSCLC CSCs). As shown in Table 1, prochlorperazine treatment also decreased the ALDH+ CL152 population in a dose-dependent manner. In conclusion, prochlorperazine showed low or minimal cytotoxic effects in NSCLC cells.
21 ± 1.4
0-2.5
0-2.5
0-2.5
0-2.5
In addition, the cytotoxicities of prochlorperazine metabolites at NSCLC parental and sphere cells were examined by SRB assay and Trypan Blue Exclusion method. It was found that N-desmethyl prochlorperazine showed better cytotoxic effect than other tested metabolites in NSCLC cell lines (see Table 2).
Also, the effect of the analogs of prochlorperazine on the clonogenicity in CL141 were examined, wherein approximately 1,000 cells were seeded in 6-well plate and treated with different analogs; after 14 days, the colonies were stained with crystal violet, photographed and counted. The results are shown in Table 3, showing that four analogs of prochlorperazine reduced clonogenicity in NSCLC cells.
Prochlorperazine induces apoptosis in CL152 spheres and synergistically enhances cytotoxicity in combine with gemcitabine.
After treatment with prochlorperazine for 48 hours, CL152 spheres were collected and analyzed by flow cytometry. Prochlorperazine led to a dose-dependent increase in apoptosis (
Prochlorperazine inhibits lung cancer cell migration and induces senescence
Cancer cells undergoing epithelial-mesenchymal transition (EMT) have been found to show increased resistance to apoptosis and certain chemotherapeutic drugs. EMT also plays a critical role in the regulation of cancer sternness. EMT is characterized by enhanced cell migration and invasion. Transwell migration assay was used to determine whether sub-cytotoxic concentration of prochlorperazine could inhibit cell motility of A549 cells in vitro. After 24 hours, treatment with 10 μM prochlorperazine significantly inhibited cell migration of A549 cells as compared with the DMSO controls (
Prochlorperazine significantly inhibits the self-renewal of NSCLC cancer spheres.
To evaluate whether the prochlorperazine alone or combined with clinical drugs, chemotherapeutic agents and EGFR-tyrosine kinase inhibitors gefitinib, treatment could suppress NSCLC cancer stem-like sphere cells in vitro. CL141 sphere cells (
In vivo examination of tumor inhibitory effects of prochlorperazine.
In the first trial (
In the drug combination test (
In other drug combination tests, CL97 (
To examine whether prochlorperazine could overcome the drug resistance, CL97 (EGFR T790M and G719A mutations) cells were subcutaneously injected into the right flank of NOD/SCID mice (female, 4-6 weeks old). One week post tumor injection, mice were randomly divided into different groups: control group (DMSO) vehicle) and prochlorperazine treatment group (5 mg/kg), target-therapy group (100 mg/kg gefitinib), and combination group (100 mg/kg gefitinib combined with 5 mg/kg prochlorperazine). In
Clinical Cases After Prochlorperazine Treatment
To prove the concept of the invention, clinical observation study of prochlorperazine (label as drug P in the figures) was performed (see
It can be concluded that prochlorperazine in combination with a chemotherapy might provide treatment benefits to patients with various cancers, particularly to overcome the drug resistant properties and to prevent cancer metastasis.
It is believed that a person of ordinary knowledge in the art where the present invention belongs can utilize the present invention to its broadest scope based on the descriptions herein with no need of further illustration. Therefore, the descriptions and claims as provided should be understood as of demonstrative purpose instead of limitative in any way to the scope of the present invention.
This application claims priority to U.S. Provisional Patent Appl. No. 62/006,630, filed on Jun. 2, 2014, the contents of which is incorporated by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2015/000380 | 6/2/2015 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62006630 | Jun 2014 | US |
