SN38 derivative folic acid liposome as well as preparation method and application thereof

Abstract

The invention provides a preparation method of folic acid modified SN38 derivative lipidosome and application of the folic acid modified SN38 derivative lipidosome in tumor treatment. A blank folic acid liposome containing glutathione (GSH) is prepared through a film ultrasonic method, and an SN38 derivative solution and the blank folic acid liposome are mixed and incubated to obtain the SN38 derivative folic acid liposome. The SN38 folic acid lipidosome prepared by the invention is of a spherical vesicle structure, and is regular in form and free of rupture; the average particle size is 118 nm, and the Zeta potential is-25 mV; and good stability and obvious slow release performance are realized. The SN38 folic acid lipid disclosed by the invention shows enhanced cytotoxicity and cellular uptake, which proves that the SN38 folic acid lipid has enhanced tumor targeting ability. Both the SN38 folic acid liposome and the SN38-Atba folic acid liposome disclosed by the invention can generate a remarkable growth inhibition effect on tumors, and the treatment effect of the SN38 folic acid liposome and the SN38-Atba folic acid liposome is obviously superior to that of the SN38 liposome and a first-line chemotherapeutic drug irinotecan clinically used for treating the tumors. Therefore, the invention provides a new treatment strategy for tumors, and has very important clinical significance and social significance.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical preparations, and in particular relates to SN38 derivative folic acid liposomes and a preparation method and application thereof. Background Art

[0002] Liposomes are bilayer vesicles formed by the directional arrangement of phospholipid bimolecules. Compared with traditional drug delivery systems, liposomes have the characteristics of improving drug efficacy, reducing adverse drug reactions and targeting effects. The advantages of liposomes as a wide range of anti-tumor drug carriers have been widely recognized. At present, the FDA has approved the marketing of a variety of anti-tumor drug liposomes, such as doxorubicin hydrochloride liposomes, daunorubicin citrate liposomes, mivastatin liposomes, cytarabine liposomes, etc. These preparations not only exert the anti-tumor efficacy of drugs in clinical practice, but also reduce the toxic side effects of drugs on normal tissues, thus showing the unique advantages of liposomes as anti-tumor drug carriers.

[0003] In the drug treatment of tumors, camptothecin derivatives are an important source of many natural anti-tumor drugs, and 7-ethyl-10-hydroxycamptothecin (SN38) is one of the most active compounds among camptothecin derivatives, which exerts anti-tumor activity by inhibiting topoisomerase I. However, the instability of the lactone ring and water insolubility of SN38 limit its clinical application to a certain extent. For this reason, the SN38 preparations that have been developed clinically are SN38's water-soluble prodrug irinotecan and irinotecan liposomes, which are first-line chemotherapy drugs for neoadjuvant chemotherapy, adjuvant chemotherapy and synchronous chemoradiotherapy of tumors.

[0004] Although existing SN38 drugs have played a huge role in clinical anti-tumor treatment, most cancer patients still suffer from a large number of serious side effects when using these drugs due to their lack of tumor targeting. In addition, existing SN38 liposomes also have problems such as low drug loading, low encapsulation rate, and poor efficacy.

[0005] In order to solve the above problems, on the one hand, the present invention modifies the structure of SN38 to prepare a more hydrophobic SN38 derivative, and then encapsulates it in liposomes through the GSH gradient active drug loading method to increase its drug loading and encapsulation rate. On the other hand, folate receptors are overexpressed on the surface of various tumor cells, but their expression in most normal tissues is relatively low, which enables folate-modified liposomes to specifically and actively target tumor cells, allowing drugs to selectively enter tumor cells, better reduce drug toxicity and side effects and improve drug efficacy. Therefore, the present invention uses folate-modified liposomes to efficiently deliver SN38 derivatives, further enhancing their targeting and improving efficacy. Summary of the invention

[0006] In view of the problems and shortcomings of the prior art, the present invention provides a folic acid liposome of an SN38 derivative and a preparation method and application thereof, so as to solve the problems in the prior art of using SN38 drugs to treat tumors, such as poor effect and easy generation of a large number of serious adverse reactions.

[0007] In order to solve the above technical problems, the present invention is implemented through the following technical solutions.

[0008] The first aspect of the present invention provides a SN38 derivative folic acid liposome for treating tumors, comprising a SN38 derivative, HSPC, cholesterol, DSPE-PEG2000, DSPE-PEG-FA, and GSH.

[0009] The SN38 derivative is SN38-MAL (molecular formula C 32 H 29 N3O 11 ) or SN38-Atba-MAL (molecular formula C 42 H 37 N3O 15 ), whose structural formula is shown below:

[0010]

[0011] The molar ratio of HSPC, cholesterol, DSPE-PEG2000 and DSPE-PEG-FA is 50-60:40-50:1-5:0.1-5; preferably, the molar ratio of HSPC, cholesterol, DSPE-PEG2000 and DSPE-PEG-FA is 50:45:4.5:0.5.

[0012] The DSPE-PEG-FA is DSPE-PEG1000-FA, DSPE-PEG 2000-FA, DSPE-PEG3400-FA, DSPE-PEG5000-FA, DSPE-PEG10000-FA, preferably DSPE-PEG3400-FA.

[0013] The second aspect of the present invention provides a method for preparing the above-mentioned SN38 derivative folic acid liposomes, comprising the following steps:

[0014] (1) HSPC, cholesterol, DSPE-PEG2000, and DSPE-PEG3400-FA are mixed and dissolved in an organic solvent to obtain a mixed solution; the mixed solution is placed in a container and prepared under reduced pressure to obtain a phospholipid film; a GSH solution is then added to hydrate the film to obtain a liposome solution; and the liposome solution is obtained by ultrasonication of a cell disruptor and filtration with a 0.22 μm microporous membrane.

[0015] (2) By dialysis, ultrafiltration or gel column chromatography, GSH in the aqueous phase outside the folate liposomes is removed to establish a GSH concentration gradient inside and outside the membrane, thereby obtaining blank folate liposomes containing GSH in the aqueous phase inside the membrane.

[0016] (3) The drug solution of SN38-MAL or SN38-Atba-MAL and blank folic acid liposomes were stirred and incubated at 60° C. to obtain active drug-loaded folic acid liposomes, and the organic solvent was removed to obtain SN38 folic acid liposomes or SN38-Atba folic acid liposomes.

[0017] The organic solvent in step (1) is selected from one or more of chloroform, dichloromethane and methanol, preferably chloroform.

[0018] The cell disruptor ultrasonic treatment time in step (1) is 2-10 min and the power is 60-200 W; preferably, the ultrasonic treatment time is 5 min and the power is 120 W.

[0019] The drug solution in step (3) is one or more of acetonitrile, methanol, ethanol, acetone, dimethyl sulfoxide and N,N-dimethylformamide, preferably acetonitrile and dimethyl sulfoxide (9:1, v:v), and the amount of the organic solvent can be 1%-20%, preferably 5%-10%.

[0020] The third aspect of the present invention provides the use of the above-mentioned SN38 derivative folic acid liposome in the preparation of drugs for preventing or treating tumors.

[0021] The tumor is selected from one or more of colon cancer, liver cancer, lung cancer, gastric cancer, pancreatic cancer, rectal cancer, cervical cancer, breast cancer, ovarian cancer, thyroid cancer, melanoma, and lymphoma; preferably colon cancer.

[0022] Compared with the prior art, the present invention has the following technical effects:

[0023] (1) The present invention verifies through in vitro cell experiments that SN38 derivative folic acid liposomes can increase the uptake of colon cancer cells and effectively inhibit the growth of colon cancer cells, and the effect is significantly better than that of SN38 single compound and SN38 ordinary liposomes.

[0024] (2) The present invention verifies through animal experiments that the SN38 derivative folic acid liposome can effectively inhibit the growth process of tumors in vivo, and the therapeutic effect is significantly better than irinotecan, a drug recommended for the first-line chemotherapy regimen for the clinical treatment of tumors. It can be used as a new treatment regimen for tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is the SN38-MAL in Example 1 1 H-NMR spectrum;

[0026] Figure 2 is the SN38-Atba-MAL in Example 2 1 H-NMR spectrum;

[0027] Figure 3 This is a display of SN38 folic acid liposomes in Example 2 under a transmission electron microscope;

[0028] Figure 4 This is a schematic diagram of the particle size detection results of SN38 folic acid liposomes in Example 2;

[0029] Figure 5 This is a schematic diagram of the Zeta potential detection results of SN38 folic acid liposomes in Example 2;

[0030] Figure 6 This is a schematic diagram of the stability test results of SN38 folic acid liposomes in Example 3;

[0031] Figure 7 This is a schematic diagram of the drug release curve of SN38 folic acid liposomes in rat plasma in Example 4;

[0032] Figure 8 This is a schematic diagram of the cytotoxicity results of SN38 folic acid liposomes in Example 5;

[0033] Fig. 9 This is a schematic diagram of the cellular uptake results of SN38 folic acid liposomes in Example 6;

[0034] Fig.10 This is a schematic diagram of the results of the changes in tumor volume in mice in Example 7;

[0035] Fig.11 This is the anatomical appearance of the mouse tumor in Example 7;

[0036] Fig.12 This is a schematic diagram of the mouse tumor weight results in Example 7.

[0037] Fig.13 This is a schematic diagram of the results of tumor volume changes in mice in Example 8. DETAILED DESCRIPTION

[0038] Example 1: Synthesis of SN38-MAL

[0039] Dissolve 1g of 3-maleimidopropionic acid in 20ml of dichloromethane, add 1.0eq of dicyclohexylcarbodiimide, react at room temperature for 5 minutes, add excess ethylene glycol and catalytic amount of DMAP (about 0.05eq), react at room temperature for 2 hours. After the reaction is completed, spin dry the reaction solution, add 20ml of ethyl acetate, filter, and separate the filtrate using a silica gel plate (dichloromethane: methanol (volume ratio 20:1)) to obtain the intermediate product. Weigh 100mg of the intermediate product, dissolve it in anhydrous dichloromethane, add 0.66eq triphosgene and 4.5eq DMAP under nitrogen protection, react for 5min, add 1.0eq SN38, and react at room temperature for 0.5h. The reaction solution was washed with 5% by weight saturated citric acid solution and saturated NaCl solution, and the organic phase was added with anhydrous Na2SO4 and filtered. The filtrate was separated using a silica gel plate (dichloromethane: methanol (volume ratio 20:1)) to obtain SN38-MAL. The product was identified by nuclear magnetic resonance spectroscopy ( Figure 1 ), confirming the successful synthesis.

[0040]

[0041] Example 2: Synthesis of SN38-Atba-MAL

[0042] Dissolve 100 mg SN38 in 20 mL of anhydrous dichloromethane, add 1.2 eq di-tert-butyl dicarbonate (boc) and 1.5 eq pyridine under stirring, and react at room temperature until the solution is clear and transparent. After the reaction is completed, wash the reaction solution with 5% by weight saturated citric acid solution and saturated NaCl in turn, take the organic phase and remove water with anhydrous Na2SO4, and evaporate the organic solvent to obtain SN38-boc for use. Dissolve 100 mg 4-(acetoxy)benzyl alcohol (Atba) in 20 mL of anhydrous dichloromethane, add 0.66 eq triphosgene and 4.5 eq DMAP under nitrogen protection; after reacting for 5 minutes, add 0.5 eq SN38-boc, and react at room temperature for 4 hours. After the reaction is completed, the reaction solution is washed with 5% by weight saturated citric acid solution and saturated NaCl in turn, the organic phase is dehydrated with anhydrous Na2SO4, filtered, and the filtrate is separated by a silica gel plate (dichloromethane: methanol (volume ratio 20:1)) to obtain SN38-boc-Atba. 100 mg SN38-boc-Atba is dissolved in an appropriate amount of dichloromethane, 10% (v / v) trifluoroacetic acid is added under stirring, and after reacting at room temperature for 3 hours, dichloromethane is repeatedly added to the reaction solution and dried until the trifluoroacetic acid is completely removed, and SN38-Atba is obtained for standby use. 100 mg of the intermediate product in Example 1 is dissolved in 20 mL of anhydrous dichloromethane, 0.66 eq triphosgene and 4.5 eq DMAP are added under nitrogen protection, and after reacting for 5 minutes, 1 eq SN38-Atba is added and reacted at room temperature for 0.5 hours. After the reaction, the reaction solution was washed with 5% by weight saturated citric acid solution and saturated NaCl solution, and the organic phase was dehydrated with anhydrous Na2SO4 and filtered. The filtrate was separated using a silica gel plate (dichloromethane: methanol (volume ratio 20:1)) to obtain SN38-Atba-MAL. The product was identified by nuclear magnetic resonance spectroscopy ( Figure 2 ), confirming the successful synthesis.

[0043]

[0044] Example 3: Preparation of SN38 derivative folic acid liposomes

[0045] Preparation of blank folic acid liposomes: 42mg HSPC, 20mg Chol, 12mg DSPE-MPEG2000, 3mg DSPE-MPEG3400-FA were mixed and dissolved in 10mL chloroform to obtain a mixed solution; 600mg glutathione (GSH) was dissolved in 5ml pure water to prepare a 400mM GSH solution. The mixed solution was placed in a round-bottom flask and prepared under reduced pressure to obtain a phospholipid film; then the above 5mL GSH solution was added to the film for hydration treatment for 10min to obtain a crude folic acid liposome solution; the folic acid liposome was obtained by ultrasonication of a cell disruptor for 5min and filtration with a 0.22μm microporous membrane. The obtained folic acid liposomes were transferred to a dialysis bag with a molecular weight cutoff of 3000 and dialyzed in a dialysate of 150 mM NaCl and 1 mM EDTA-2Na at 38°C to remove extraliposome GSH; the dialysate was replaced every 2 hours for a total of 8 times to obtain blank folic acid liposomes encapsulating GSH.

[0046] Preparation of drug-loaded liposomes: 1 mg SN38-MAL or SN38-Atba-MAL was dissolved in 0.1 mL of a mixed solvent of acetonitrile and DMSO (9:1, v:v) to a final concentration of 10 mg / mL. Under vigorous stirring, the SN38-MAL or SN38-Atba-MAL solution was dispersed in 0.3 mL of 150 mM NaCl solution, and then 1 mL of the blank folic acid liposomes was added and incubated at 60°C for 20 min to complete the drug encapsulation. After encapsulation, it was placed in a 4°C refrigerator for storage for later use.

[0047] The physicochemical characteristics of the SN38 folic acid liposomes prepared by the above method were detected and characterized. Figure 3-6 As shown. Figure 3 The particle morphology of SN38 folic acid liposomes under a transmission electron microscope is shown, which presents a spherical or quasi-spherical vesicle structure with regular morphology and no cracks. Figure 4 The particle size of SN38 folic acid liposomes (measured by Malvern particle size analyzer) was displayed. The results showed that the average particle size was 118 nm, the particle size range was narrow, the particle size distribution was uniform, and it showed a normal distribution. Figure 5 The Zeta potential test result of SN38 folic acid liposomes was -25mV. Furthermore, the obtained SN38 folic acid liposomes were placed at 4 degrees for 28 days, during which the particle size change was monitored to investigate their stability. The results showed that the particle size of SN38 folic acid liposomes tended to be relatively stable, indicating that the SN38 folic acid liposomes prepared by the present invention had good stability (see Figure 6 ).

[0048] Example 4: Drug release from SN38 folic acid liposomes

[0049] Take 2.5 μL SN38 folic acid drug-loaded liposomes, add 50 μL rat plasma, mix well and incubate at 38°C, add 200 μL 1% acetic acid methanol at different time points, vortex for 1 minute, extract in water bath ultrasound for 10 minutes, centrifuge and take the supernatant, perform HPLC analysis, determine the content of SN38-GSH (i.e., the reaction product of SN38-MAL and GSH) and SN38 original drug, calculate the drug release rate, and draw the drug release curve. The results are as follows Figure 7 As shown, the drug release rate of SN38 folic acid drug-loaded liposomes is slow, with only 66% of the drug released within 48 hours, indicating that the SN38 folic acid drug-loaded liposomes have obvious sustained-release properties.

[0050] Example 5: Cytotoxicity of SN38 folic acid liposomes

[0051] Mouse colorectal cancer cells (CT26 cells) in the logarithmic growth phase were digested with trypsin, centrifuged, and concentrated to 5 × 10 3 Cells / well were seeded in a 96-well plate and incubated overnight; then different concentration gradients of free SN38, SN38-Lip or FA-SN38-Lip were added and the cells were incubated for 48 hours; then 10 μL of MTT solution (5 mg / mL) was added to each well, and the plate was incubated for another 4 hours; finally, the culture medium in each well was replaced with 100 μL of DMSO, and the absorbance was measured at a wavelength of 570 nm to draw a cell survival rate curve. The results are shown in Figure 8 As shown, due to the presence of the folate target head, FA-SN38-Lip showed strong cytotoxicity on the growth of CT 26 cells, which was about 4 times stronger than that of the SN38-Lip group, indicating that FA-SN38-Lip has enhanced tumor targeting ability.

[0052] Example 6: Cellular Uptake of SN38 Folate Liposomes

[0053] Mouse colorectal cancer cells (CT26 cells) in the logarithmic growth phase were digested with trypsin, centrifuged, and concentrated to 3 × 10 5 Cells / well were inoculated in a 12-well plate and incubated overnight; then 1 μg / mL (converted to SN38 concentration) of free SN38, SN38-Lip or FA-SN38-Lip was added and the cells were incubated for 24 hours; then the cells were washed 3 times with PBS, and 400 μL of 1% acetic acid methanol was added to each well. After the cells were transferred to EP tubes, they were extracted by water bath ultrasound for 10 minutes, centrifuged and the supernatant was taken for HPLC analysis to determine the content of SN38-GSH and draw a drug content curve. The results are shown in Fig. 9As shown, the content of SN38-GSH in FA-SN38-Lip was about 14 times that of the SN38-Lip group, indicating that due to the presence of the folate target head, tumor cells increased their uptake of the FA-SN38-Lip group.

[0054] Example 7: Pharmacodynamics test of SN38 folic acid liposomes

[0055] CT26 cells in the logarithmic growth phase were digested with trypsin, centrifuged, and resuspended in PBS. 100 μL of the cells were pipetted to a concentration of 2×10 6 The cell suspension was inoculated subcutaneously on the back of male Balb / C mice to establish a mouse colon cancer tumor model. 3 At about 14:00 p.m., the tumor-bearing mice were divided into 4 groups according to the size of the tumor, with 6 mice in each group, and were injected with normal saline (blank control), irinotecan solution (CPT-11, 35 mg / kg), SN38-Lip (9 mg / kg, calculated as SN38) and FA-SN38-Lip (9 mg / kg, calculated as SN38) through the tail vein. The drugs were administered once every 6 days for a total of 4 times, and the tumor volume of the mice was measured every 2 days. The tumor volume of the mice = (long diameter of the tumor × short diameter of the tumor) 2 ) / 2. When the tumor volume of the blank control group reached 2000mm 3 At 4 pm, all mice were killed, and the tumors were removed and weighed.

[0056] The results are as follows Figure 10-12 As shown, Fig.10 The tumor volume in each group of mice after treatment was shown. Fig.11 The schematic diagram of the anatomical appearance of the tumors in each group of mice is shown. Fig.12 The results show that the tumor mass of each group of mice after treatment was completed. The results showed that irinotecan solution (35 mg / kg) had no anti-tumor activity; while the tumor volume and weight of the FA-SN38-Lip group were the smallest among all groups. In contrast, the FA-SN38-Lip group showed more effective anti-tumor activity against CT26 colorectal tumors than SN38-Lip.

[0057] Example 8: Pharmacodynamics test of SN38-Atba folic acid liposomes

[0058] CT26 cells in the logarithmic growth phase were digested with trypsin, centrifuged, and resuspended in PBS. 100 μL of the cells were pipetted to a concentration of 2×10 6 The cell suspension was inoculated subcutaneously on the back of male Balb / C mice to establish a mouse colon cancer tumor model. 3At about 14:00, the tumor-bearing mice were divided into 4 groups according to the size of the tumor, with 6 mice in each group, and physiological saline (blank control), irinotecan solution (CPT-11, 35 mg / kg), SN38-Lip (9 mg / kg, calculated as SN38) and FA-SN38-Atba-Lip (9 mg / kg, calculated as SN38) were injected into the tail vein. The drugs were administered once every 6 days, for a total of 3 times, and the tumor volume of the mice was measured every 2 days. The tumor volume of the mice = (long diameter of the tumor × short diameter of the tumor) 2 ) / 2. After three administrations, all mice were killed. The changes in tumor volume in each group of mice are shown in Fig.13 As shown, irinotecan solution (35 mg / kg) had no obvious anti-tumor activity. In contrast, the FA-SN38-Atba-Lip group showed more effective anti-tumor activity against CT26 colorectal tumors than SN38-Lip. It can be clearly seen from the above results that the SN38-Atba folic acid liposomes of the present invention can produce a significant growth inhibitory effect on tumors, and its effect is significantly better than irinotecan, a first-line treatment recommended drug for clinical treatment of tumors. In this regard, the present invention provides a new treatment strategy for tumors, which can improve the current situation that irinotecan, a first-line treatment drug for tumors, has poor effects and is prone to a large number of adverse reactions. The present invention plays an important role in improving the therapeutic effect and prognosis of tumors, and has very important clinical and social significance.

[0059] The above specific implementation method part specifically introduces the analytical method involved in the present invention. It should be noted that the above introduction is only to help those skilled in the art better understand the method and ideas of the present invention, rather than limiting the relevant content. Without departing from the principle of the present invention, those skilled in the art may also make appropriate adjustments or modifications to the present invention, and the above adjustments and modifications shall also fall within the scope of protection of the present invention.

Claims

1. A SN38 derivative folic acid liposome for preventing and / or treating tumors, characterized in that: Including SN38 derivatives, HSPC, cholesterol, DSPE-PEG2000, DSPE-PEG-FA, and glutathione.

2. The SN38 derivative according to claim 1, characterized in that: The SN38 derivative is SN38-MAL or SN38-Atba-MAL, and its structural formula is as follows:

3. The folic acid liposome according to claim 1, characterized in that The molar ratio of the HSPC, cholesterol, DSPE-PEG2000 and DSPE-PEG-FA is 50-60:40-50:1-5:0.1-5.

4. The folic acid liposome according to claim 1 or 3, characterized in that The DSPE-PEG-FA is DSPE-PEG1000-FA, DSPE-PEG 2000-FA, DSPE-PEG3400-FA, DSPE-PEG5000-FA, DSPE-PEG10000-FA, etc., preferably DSPE-PEG3400-FA.

5. The SN38 derivative folic acid liposome for preventing and / or treating tumors according to claim 1, characterized in that: The tumor is selected from solid tumors.

6. The method for preparing SN38 derivative folic acid liposomes for preventing and / or treating tumors according to any one of claims 1 to 5, characterized in that: The steps include: (1) HSPC, cholesterol, DSPE-PEG2000, and DSPE-PEG3400-FA are mixed and dissolved in an organic solvent to obtain a mixed solution; the mixed solution is placed in a container and prepared under reduced pressure to obtain a phospholipid film; a glutathione (GSH) solution is then added to hydrate the film to obtain a liposome solution; and the liposome solution is filtered through a cell disruptor ultrasound and a microporous membrane to obtain folic acid liposomes. (2) By dialysis, ultrafiltration or gel column chromatography, GSH in the aqueous phase outside the folate liposomes is removed to establish a GSH concentration gradient inside and outside the membrane, thereby obtaining blank folate liposomes containing GSH in the aqueous phase inside the membrane. (3) The drug solution of SN38-MAL or SN38-Atba-MAL and blank folic acid liposomes were stirred and incubated at 60° C. to obtain active drug-loaded folic acid liposomes, and the organic solvent was removed to obtain SN38 folic acid liposomes or SN38-Atba folic acid liposomes.

7. The preparation method according to claim 6, characterized in that: The organic solvent in step (1) is selected from one or more of chloroform, dichloromethane and methanol, preferably chloroform.

8. The preparation method according to claim 6, characterized in that: The drug solution in step (3) can be one or more of acetonitrile, methanol, ethanol, acetone, dimethyl sulfoxide and N,N-dimethylformamide, preferably acetonitrile and dimethyl sulfoxide (9:1, v:v), and the amount of the organic solvent can be 1%-20%, preferably 5%-10%.

9. Use of the SN38 derivative folic acid liposome according to any one of claims 1 to 5 and / or the SN38 derivative folic acid liposome prepared according to the preparation method according to any one of claims 6 to 8 in the preparation of drugs for preventing or treating tumors.

10. The use according to claim 9, characterized in that: The tumor is selected from one or more of colon cancer, liver cancer, lung cancer, gastric cancer, pancreatic cancer, rectal cancer, cervical cancer, breast cancer, ovarian cancer, thyroid cancer, melanoma, and lymphoma, preferably colon cancer.

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