Patent Application: US-201414285651-A

Abstract:
the present invention discloses a method of preventing and treating gefitinib - resistant non - small - cell lung cancer , comprising administering an effective amount of an alkaloid . a pharmaceutical composition comprising an alkaloid admixed with a pharmaceutical carrier for treating gefitinib - resistant non - small - cell lung cancer is also disclosed therein .

Description:
sanguinarine , which is a natural benzophenanthridine alkaloid , is isolated from chinese medicinal herb macleaya cordata and also from north american herb sanguinaria canadensis . as the major constituent , the pharmacological effects of sanguinarine have been widely studied in many fields for a long time , for example , as an anti - microbe agent [ 9 , 10 ], an anti - inflammation agent [ 11 , 12 ] and an anti - oxidation agent [ 13 , 14 ]. it is also approved by the fda as an antibacterial or antiplaque agent in toothpastes in 2003 [ 15 ]. however , its effect on nsclc and especially on gefitinib - resistant nsclc has never been studied and thus not readily known to one skilled in the art . in the instant invention , it is the first time to report the anticancer effect of sanguinarine on gefitinib - resistant nsclc , which may provide hopes to cancer patients who have developed gefitinib resistance . sanguinarine powder was purchased from sigma aldrich . antibodies of bcl - 2 , bax and bcl - xl were purchased from santa cruz biotechnology while antibodies of phospho - p38 , phosphor - jnk , phospho - akt , total - egfr , phosphor - egfr y1045 were purchased from cell signaling technology . four nsclc cell lines , h1975 ( egfr l858r + t790m ), h1650 ( egfr exon19 deletion ), h2228 ( eml4 - alk fusion gene ) and a549 ( egfr wild type ) were purchased from atcc . all cell lines were cultivated with rpmi 1640 medium supplemented with 10 % fetal bovine serum ( gibco ), 100 u / ml penicillin and 100 μg / ml streptomycin ( gibco ). all cells were cultivated at 37 ° c . in an incubator supplying with 5 % co 2 . nsclc cells were seeded on a 96 - well microplate at a confluence of 4000 cells / well , and were cultured overnight to allow cell adhesion . wide concentration of range of sanguinarine was added into the wells and the plate was incubated for 24 or 48 hrs with vehicle ( dmso ) as the control . each dosage was repeated in triplicate . 10 μl of mtt ( 5 mg / ml ) was added to each well and the plate was placed back into the incubator for another 4 hrs to allow the entry of mtt into the cells . then 100 μl of the resolved solution ( 10 % sds and 0 . 01 % m hcl ) was added to each well and the plate was further incubated at 37 ° c . for 4 hrs until the formazan crystals had dissolved . finally , the absorbance of the plate was measured at 570 nm ( absorbance ) and 650 nm ( reference ) by a microplate reader ( tecan ). the percentage of cell viability is calculated as the percentage change of the absorbance of treated cells to the untreated cells . assessment of apoptosis levels by annexin v / pi staining with flow cytometry annexin v / pi staining is used to detect early and late stages of apoptosis . according to the manufacturer &# 39 ; s protocol ( bd pharmingen ), 5 × 10 5 cells of the treated and control groups were harvested , washed , and double - stained with annexin v / pi for 15 min at room temperature in dark . then apoptotic cells were quantitatively counted by a flow cytometer ( bd facsaria iii ). early stage of apoptotic cells were stained with annexin v - positive and pi - negative , while the late stage of apoptotic cells were stained with annexin v - positive and pi - positive . cells were lysed in ripa lysis buffer with protease and phosphatase inhibitors added for total whole cell protein extraction . the concentration of the total protein extract was determined by bio - rad dctm protein assay kit ( bio - rad ). then samples were loaded and separated onto a 10 % sds - page gel and then the separated proteins were transferred to a nitrocellulose ( nc ) membrane . membranes were blocked with 5 % milk without fat in tbst for 1 hour at room temperature . primary antibodies ( 1 : 1000 dilutions for cell signaling antibodies ; 1 : 500 dilutions for santa cruz antibodies ) were added and incubated overnight at 4 ° c . on the other day , membrane was washed by tbst for three times ( 5 mins / wash ), while secondary fluorescent antibody ( 1 : 10000 dilutions ) was added to membrane and incubated at room temperature for 1 hr . actin was used as loading control and for normalization . the signal intensity of the membranes was detected by odessy ( li - cor ). levels of reactive oxygen species ( ros ) generation was analyzed by using fluorescent probe dichlorofluorescein diacetate ( dcfda ) staining , which is a specific superoxide tracing dye . cells were pretreated with dcfda at working concentration of 20 μm ( abcam ) for 30 min at 37 ° c . prior to sanguinarine treatment . then cells were incubated with sanguinarine and vehicle control for 30 mins , 1 hr and 2 hrs , and then were harvested and re - suspended in pbs . fluorescence signal was measured with flow cytometer with excitation and emission settings of 488 and 525 nm , respectively . all of the data is expressed as mean ± sem of three individual experiments . differences between groups were determined by one way analysis of variance ( anova ), with p values & lt ; 0 . 05 was considered as significant . study of sanguinarine against four nsclc cell lines after mtt cytotoxicity assay this example described the cytotoxicity effect induced by sanguinarine in four nsclc cell lines utilizing mtt cytotoxicity assay . among the four cell lines , a549 is wild type egfr , while the other three are gefitinib - resistant nsclc lines with different mutations . h2228 contains eml4 - alk fusion gene mutation and could be used as egfr survival independent control cells . ic 50 values of each cell line after a 24 hr treatment are shown in table 1 . conclusion : from the results shown in table 1 , sanguinarine has cytotoxicity effect in human nsclc cell lines , especially in h1975 ( egfr l858r + t790m ); while it shows relatively low cytotoxicity effect in a549 ( egfr wild type ) cell lines . thus , sanguinarine is shown to be effective in treating gefitinib - resistant non - small - cell lung cancer . western blot analysis of the protein level of egfr after sanguinarine treatment in example 3 , the protein level of egfr after sanguinarine treatment with difference dosage and duration was analyzed . fig1 a shows the results of western blot analysis of the protein level of egfr after treating with sanguinarine of different concentrations for 24 hrs . fig1 b shows the results of western blot analysis of the protein level of egfr after treating with sanguinarine at 1 . 25 μm for 0 - 24 hrs . conclusion : the results indicate that sanguinarine induced egfr degrades in a dose - dependent and time - dependent manner . example 4 further studies the sanguinarine - induced apoptosis in gefitinib - resistant nsclc cell line h1975 ( egfr l858r + t790m ) upon treatment with sanguinarine at a concentration of 1 . 25 μm for 24 hrs , significant induction of apoptosis in h1975 cells was observed , as shown in fig2 . as shown in fig2 a , intact cells were gated in the same area , and the treatment of sanguinarine resulted in 20 % more cell debris . in fig2 b , the percentages of both early stage ( q3 ) and late stage ( q2 ) apoptotic cells were quantitatively counted by flow cytometer , and the result shows that there is a significant 7 . 2 - fold increase in early stage of apoptosis of the sanguinarine - treatment group as compared with the control group . conclusion : the results indicate that sanguinarine induces apoptosis in gefitinib - resistant nsclc cell line h1975 significantly . study of sanguinarine - induced apoptosis in h1975 cells via mitochondria dependent pathway example 5 analyzed the protein level of bcl - 2 , bcl - xl and bax after sanguinarine treatment with difference dosages for 24 hours . conclusion : from the results shown in fig3 , sanguinarine is shown to remarkably suppress the expression of bcl - 2 , bcl - xl , and slightly up - regulate bax , which will up - regulate the ratio of bax / bcl - 2 , leading to the disruption of mitochondrial membrane integrity and further leading to significant apoptosis in gefitinib - resistant nsclc cell line h1975 . example 6 further studies if the sanguinarine - induced apoptosis in h1975 ( egfr l858r + t790m ) is in caspases - dependent manner h1975 cells were treated with 1 . 25 μm sanguinarine and 1 . 25 μm sanguinarine together with 50 μm pan - caspase inhibitor for 24 hrs . as shown in fig4 a , intact cells were gated in the same area , and the treatment of 1 . 25 μm sanguinarine together with 50 μm pan - caspase inhibitor resulted in 20 % less cell debris compared with the group treated with 1 . 25 μm sanguinarine only . as shown in fig4 b , the percentages of both early stage ( q3 ) and late stage ( q2 ) apoptotic cells were quantitatively counted by flow cytometer , and the result shows that the percentages were decreased about 3 - fold in both early and late stages of apoptosis of the sanguinarine & amp ; pan - caspase inhibitor treatment group compared with the sanguinarine group . conclusion : the results show that pan - caspase inhibitors can attenuate sanguinarine - induced apoptosis in h1975 cells ; in other words , caspases activation is necessary for the apoptosis induced by sanguinarine . study on reactive oxygen species ( ros ) generation in sanguinarine - induced apoptosis in h1975 cells at 30 minutes after 1 . 25 μm sanguinarine treatment , ros was greatly accumulated and the ros intensity was shown in fig5 a . ros intensity was increased with the increase of sanguinarine treatment time . sanguinarine - induced apoptosis in h1975 cells , in which the cells were pre - treated with 5 μm nac and a ros generation inhibitor , was also studied . as shown in fig5 b , it can be observed that apoptosis induced by sanguinarine was completely blocked . further , the protein levels of jnk , p38 and egfr after the treatment of 5 μm nac and / or 1 . 25 μm sanguinarine were shown in fig6 , indicating that the ros generation inhibitor can also block the phosphorylation of jnk , p38 and egfr degradation . also , p - egfr 1045 as shown in fig6 is a tyrosine ( y ) phosphorylation site of egfr at amino acid position 1045 ( y1045 ) and phosphorylation of this site will lead to activation of egfr degradation after cbl docking to this site conclusion : the results of this study show that ros generation is essential for sanguinarine - induced apoptosis in h1975 cells and required at the early stage of the apoptosis . this is the first study demonstrating that sanguinarine exerts remarkable cytotoxic effect on killing gefitinib - resistant nsclc cells . it is also the first report showing that sanguinarine can effectively trigger degradation of egfr . the results show that sanguinarine has potency of anticancer effect by potentiating egfr degradation , mapk ( jnk and p38 ) activation , mitochondria disruption , and caspases activation . taken together , these results indicate that sanguinarine could be used as a candidate agent against gefitinib - resistant nsclc patients , especially for the group of patients with egfr l58r + t790m mutation which represents 49 % of all gefitinib resistance cases . the exemplary embodiments of the present invention are thus fully described . although the description referred to particular embodiments , it will be clear to one skilled in the art that the present invention may be practiced with variation of these specific details . hence this invention should not be construed as limited to the embodiments set forth herein . 1 . jemal , a ., et al ., cancer statistics , 2010 . ca : a cancer journal for clinicians , 2010 . 60 ( 5 ): p . 277 - 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1 cell line . phytomedicine : international journal of phytotherapy and phytopharmacology , 2012 . 19 ( 10 ): p . 890 - 5 . 12 . niu , x ., et al ., the anti - inflammatory effects of sanguinarine and its modulation of inflammatory mediators from peritoneal macrophages . european journal of pharmacology , 2012 . 689 ( 1 - 3 ): p . 262 - 9 . 13 . vrba , j ., e . orolinova , and j . ulrichova , induction of heme oxygenase - 1 by macleaya cordata extract and its constituent sanguinarine in raw 264 . 7 cells . fitoterapia , 2012 . 83 ( 2 ): p . 329 - 35 . 14 . vrba , j ., et al ., sanguinarine is a potent inhibitor of oxidative burst in dmso - differentiated hl - 60 cells by a non - redox mechanism . chemico - biological interactions , 2004 . 147 ( 1 ): p . 35 - 47 . 15 . bai , l . p ., et al ., site - specific binding of chelerythrine and sanguinarine to single pyrimidine bulges in hairpin dna . analytical and bioanalytical chemistry , 2008 . 392 ( 4 ): p . 709 - 16 .