咯萘啶在制备抗肿瘤药物和逆转肿瘤多药耐药性的药物中的应用
The invention relates to the new use of malaridine, in particular to malaridine in preparing antitumor drug and reverse multi-drug resistance of tumor application of the medicament. High mortality rate of tumor patients, wherein one of the important factors to be ignored is to generate tumor drug resistance, so that the patient is not sensitive to the chemotherapy drugs, chemotherapy failure result. The inventor of the application has for many years been committed to reverse the tumor multi-drug resistance (multidrug resistent, MDR) research, it has been found that with a different degree of reverse activity there are a dozen or so ways, such as fills the age to decide , quinine, [...] , interferon (INF-a), psoralene, isopsoralen, Matrine, oxymatrine, and tetrandrine, and so on, but reverse active are not and have been used for clinical reversal of MDR [...] cyclosporin A (CsA) and VP2 (VPL), and serious cardiovascular system toxicity and serious immunosuppressive, renal toxicity and central nervous system toxicity also hamper their clinical application. Therefore, the search for more safe and effective reversing MDR, as soon as possible and applied to the clinical is still an important issue facing US. Today's tumor tumor drug resistance is a major problem in the treatment of, scholars is also hot spots of the study at home and abroad. In the past twenty years, it has been found that tumor to the chemotherapy drug-resistant mechanism has many, wherein gene mdrl and its encoding glycoprotein PgP with the hyper-expression of MDR is closely related to a mechanism, with energy-dependent "pump" function, combined with antineoplastic, at the same time is connected to the ATP site of the polynucleotide, the energy of ATP hydrolysis of the drug released after the discharge cells, reducing the intracellular drug concentration, drug resistance. Because of the drug resistance, such as: anthracene, alkaloids (CHANGCHUN new alkali, Eagle's nest, three) are leukemia, lymphoma, and lung cancer, breast cancer the most effective solid tumor such as pharmaceutical, therefore, the application of reversal, is to overcome tumor drug resistance, improve chemotherapy effect an important means of. Currently, many pharmaceutical can be confirmed in vitro reverse MDR, and CsA and VPL has already been applied to the clinical. But serious toxic side effects limit their use. Therefore, the search for high-efficient and low-toxicity MDR reversal agent become there is an urgent need to overcome the problem. The aim of the invention is to find an anti-tumor medicine. Another purpose of this invention is to find a reverse tumor medicine multi-drug resistance. The inventors have found that antimalarial malaridine with anti-tumor and obvious role of the reversal of the MDR tumor. Malaridine (pyronaridine, PND), its chemical name 2-methoxy-7-chloro -10-[ 3, 5-bis-(pyrrolidinyl-1-methyl)-4-hydroxy phenyl-amino] benzo-1, 5-naphthyridine. To compare with earlier published in pharmacy Journal 1979 Dec; 14 (12): 736-7. This invention offers malaridine and its salt in the preparation of the application of the anti-tumor drugs. The invention further provides malaridine and its salt in preparing reverse multi-drug resistance of tumor application of the medicament. The result of the study is that to human leukaemia cells PND K562, H160 and human solid tumor cells MCF-7, KB, A549 has an inhibitory effect of cells, that have extensive PND-inhibiting activity in vitro, can be made into the anti-tumor drug. The relevant PND the multi-drug resistance of tumor cells of the research shows that: PND the drug-resistant cell is still displayed with the sensitive cell similar cytotoxicity; below IC50 doses, doxorubicin can be remarkably improved PND (Doxorubicine, ADR) to the toxic effect of drug-resistant cells, drug resistant cells to restore the sensitivity of the ADR; ADR PND a marked increase in the accumulation of the drug-resistant cells; can improve Rh123 drug resistant cells to the uptake, reduce Rh123 of the efflux; and the single ADR ADR can be improved for the percentage of inducing apoptosis. Research shows that, with PND extensive in vitro inhibiting activity and efficient multi-drug resistance of tumor cells of reverse active, can be used as used in clinical MDR reversal. Research shows that, with other anti-cancer drugs malaridine can be/radiotherapy or other biological preparation of an anti-tumor drug use; or, with other anti-cancer drug/radiotherapy or other biological preparation as reverse combination of multi-drug resistance of tumor drug use. In the prior art, K562/A02, MCF-7/ADR and KB/VCR200 cells are MDR multidrug-resistant phenotype of a cell line, HL60/ADR and A549DDP MRP phenotype of a cell is a MDR cell line. According to the result of study of this invention, prompt, even though clinical patient has produced resistance as a result of chemotherapy, PND can still be used to kill the tumor cell. Furthermore, the PND 4.40, 3 . 30, 2.20 and 1.10 the under M, reverse K562/A02 and MCF-7/ADR multiple reach 240.5, 79 . 75, 17.72, 2.26 times and 29.68, 4 . 30, 2.70, 2.84 times. It has also been discovered, can be increased in the ADR PND K562/A02 and MCF-7/ADR the accumulation of in, 2.20 the when M, the K562/A02 increase concentration of the ADR in the cell 13.24 times; further study finds that, PND Rh123 drug-resistant cells to enhance the uptake, inhibiting the efflux. Rh123 Pgp pump function indicator is, estimated from our results, glycoprotein Pgp PND may is applied, reducing the intracellular drug efflux, so as to play the role of reversing MDR; at the same time the study shows that jointly use ADR PND, the apoptosis of tumor cells can be enhanced, in 1.10, 2 . 20 and 3.3 the dosage M, the K562/A02 percentage of apoptosis by the single use of the ADR 2.64% cent to 11.69%, 19 . 14% and 46.18%, therefore, increase the drug-resistant cell apoptosis is also one of the MDR PND reversing mechanism. To sum up, inhibiting and reversing MDR in vitro PND action is relatively strong, reliable effect as reversing MDR, its mechanism of action, on the one hand, possible and inhibiting Pgp expression, or with chemotherapy drug competition and inhibit Pgp Pgp the combination of the drug efflux pump relevant to the function of; on the other hand, may be associated with increasing tumor cell apoptosis. PND as a medicament for the treatment of malaria, its clinical iv. 3-6mg/kg consumption, can even be up to 10 mg/kg, secondary calculated every day, the highest blood drug concentration can reach 50-100 subsidence g/ml, use of this invention is far higher than the maximum reverse dose in vitro of the 4.40 M, reversing MDR PND as the clinical feasibility of the; more valuable is, clinical information display, the minor adverse reaction, only a minority of patients with headache, nausea, and nervous system side effects such as skin rash, restores drug, thereby ensuring the safety of clinical use PND; our study of this invention it has also been discovered, stronger effect than MDR reversal PND VPL, display the high efficiency PND. Therefore, we believe that, may be PND the chemotherapy in clinical tumor patients play a high-efficiency, security role of reversing MDR, a MDR reversal development of the future. A route of administration of the oral (naphthyridine piece phosphate) and intravenous injection (injection malaridine phosphate) two. The following is the human tumor cells to the inventor naphthyridine anti- and its corresponding tumor drug-resistant cell, in particular drug-resistant reverse tumor activity and its mechanism of experiments. Brief description of the Figure are as follows: Figure 1 the display PND K562/A02 the influence of the concentration of the ADR; Chart 2 display PND MCF-7/ADR to the influence of the concentration of the ADR; Figure 3 the display PND MCF-7/ADR cell uptake Rh123 impact; Figure 4 the display PND MCF-7/ADR cell uptake Rh123 impact; Figure 5A and 5B display to the front and rear cells Rh123 PND the change of the concentration. Experimental example A. Material and method 1. materials 1.1 cell: K562 and K562/A02, H160 and H160/Adr, KB and KB/VCR200, A549 and A549/DDP. The conventional culture. 1.2 drug and reagent : (Shanghai sends the laboratory) malaridine (PND), adriamycin (ADR) (shenzhen ten thousand music Company limited), MTT (Sigma). 1.3 main instrument: Hitachi 650-60 ultraviolet spectrophotometer, UV-3000 fluorescence spectrophotometer, enzyme-Titertek Multiscan, flow cytometry. 2. Method 2.1 cytotoxic test: ADR, PND, and VPL in sterile physiological saline is MTT into the corresponding concentration spare. The logarithmic phase cell, inoculate in 96 kong Wei culture plate, cell number is 2 × 104/hole, the 180 l/hole), in 37 , 5% CO2, under wet conditions of the cultured in an incubator, and 12 hours later, packet dosing, each concentration is provided with three parallel holes, the negative control plus physiological saline, the volume of the end of the 200 l/hole, 68 hours later, each hole plus MTT the 20 l (5 mg/ml), culturing an incubator for 4 hours, centrifuged (2000 rpm, 10 minutes), absorbing, by adding DMSO150 l of each hole, oscillation to the precipitation is completely dissolved, at 546 nm measuring the optical density (OD) value of each hole. Inhibition is calculated according to the following formula : = inhibition [control OD value- adds the medicine group OD value]/ control OD value × 100%. IC50 reducing the survival rate of the cells to 50% of the drug dose, according to the linear regression equation. 2.2 tumor drug-resistant reverse test: method is the same. adds the medicine group are (1) ADR ; (2) ADR+PND (4.40 the M) ; (3) ADR+PND (the 3.30 M) ; (4) ADR+PND (the 2.20 M) ; (5) ADR+PND (the 1.10 M) ; (6) ADR+VPL (the 10 M), the negative control plus the volume of physiological salt water whenever the 200 l. 2.3 Fluorospectro spectrophotmetric determining the intracellular concentration of ADR: fluorescence can be generated by utilizing the characteristics of ADR, by measuring the intensity of fluorescence in the cell reaction in the cell to the height of the concentration of the ADR. Taking K562/A02 and MCF-7/ADR cells, serum-free for 1640 washing 2 times, the suspended in a serum-free 1640 liquid (5 × 105/ml), packet dosing : (1) ADR (the 2 M) ; (2) ADR +PND (the 2 M) (2.20 the M) ; (3) ADR+VPL (the 10 M), to the blank containing the cell suspension. In 37C oscillating incubation in water bath, for 30 min, 60 min, 90 min sampling, with cold physiological salt water washing 2 times, each sample plus 1 ml physiological saline, frozen in -20 the overnight in refrigerator [...] , taken out, after to be melted, fertilization-free crop seeds crushing cells using cell breaking, measuring fluorescence intensity and corresponding protein concentration, each of the samples for parallel three parts, its mean value, compared with a standard curve, calculated per mg protein concentration of the ADR. 2.3 FACSCalibur determining the intracellular Rh123: taking logarithm anagen K562/A02 and MCF-7/ADR cells, with cold physiological salt water washing 2 times, in order to non-serum 1640 adjusting into 5 × 105/ml, adding Rh123 (the 10 M) and PND (the 2 M), 37C water bath 60 min after, Rh123 cold physiological saline wash, then the sample is divided into heating PND the 4.40 M, the 2.20 M, the 1.10 M; plus VPL 10 the reversal and without M, continue to incubate 60 min, cold physiological salt water washing 2 times, flow cell instrument Rh123 in the cell concentration. 2.4 the fluorescence observation of cell microscope law Rh123: taking exponential phase cell, cold physiological salt water washing 2 times, respectively adding Rh123 (the 10 M) and Rh123 +PND (the 10 M) (2.20 the M), 37C, 30 min later, the fluorescence observed under microscope cells Rh123 and cameraphone. 2.5 FACSCalibur measuring apoptosis: taking logarithm anagen K562 and K562/A02 cell, to get 5 × 105/ml, adding ADR, and PND ADR+PND, continue to culture in yu Fuxiang , respectively in 24hr and 72hr out, using physiological salt water washing 2 times, by adding 4C, 70% ethanol fixed 24hr, centrifugal 1000rpm/min ethanol wash, plus PI (50 subsidence g/ml) dyeing 20 min, cell yu Liushi measuring apoptosis. 2.6 statistical processing: medical science statistical significance between groups for testing software POMS-03 processing. II. Results 1. PND inhibiting effect on human tumor cells: PND inhibiting effect on human tumor cells shown in table 1. Display results on various PND has an inhibitory effect of human tumor cells, wherein the human leukemia cell K562/A02, HL60, and HL60/ADR exhibits relatively solid tumor cells more strong inhibiting activity; in addition, drug-resistant cells to tumor PND still has the same sensitivity, such as a multi-drug resistant cell K562/A02, MCF -7/ADR, HL60/ADR, KB/V200 drug resistance of the ADR are 84.36, 47 . 73, 64.63 and 43.02 times, the drug resistance PND almost equal to 1 (P> 0.05). Table 1. PND the inhibition of human tumor cells (IC50 µm) 2. PND the role of reverse MDR tumor cells: The PND the reversal of the function of MDR tumor cells in table 2. Study found, ADR PND remarkably improve the drug-resistant cell K562/A02 and MCF-7/ADR sensitivity, the PND 4.40 the when M, its response to the reversal of the multiple 240.5 and 29.68 times, wherein the K562/A02 cell reversing after the ADR IC50 or even lower than-sensitive cell, a reverse effect that not only PND, there are synergies; and VPL10 the when M, reverse multiple is only 49.05 times and 8.84 times, can see VPL PND compared with the existing positive reversal, more prominent effect of reversing MDR, (P <0.05). Under the same dose, the ADR PND almost does not affect the toxic effect of sensitive cells, note the PND multidrug-resistant cell with specificity the role of reverse MDR. Table 2.PND multidrug resistant cells to the reversal of the effect * are the above-mentioned data adding or not adding reversal of the ADR IC50 (µm); data in multiple for reversing the brackets. 3.PND to the impact of the intracellular drug concentration: The PND the impact of the intracellular drug concentration in Figure 1, 2, fluorescent spectrophotometry ADR concentration found that the detection cells, drug resistant cells PND can increase the concentration of the ADR. PND group is not added, K562/A02 and MCF-7/ADR cells in the concentration of ADR 65.03 and 388.68ng/mg protein, after adding PND, in the cell to increased ADR 860.77 and 595.59ng/mg protein, in particular for K562/A02 cell reaches or exceeds the level of sensitive cells. 4.PND Rh123 to tumor cell uptake role of: Observed from the fluorescent microscope, Rh123 for (the 10 M) for the treatment of cells, 30 min later, the inner fluorescent very strong sensitive cells, the drug-resistant cell hardly display fluorescence, indicates that the drug-resistant cells Rh123 is nearly zero; PND after the addition, drug-resistant cell in a very strong fluorescence, restored to the sensitive cell level, results see photographs. PND can see a marked increase in drug-resistant tumor cells Rh123 concentration. The flow cytometry method detection cells Rh123 also support the above-mentioned results, see Figure 3, 4. 5.PND the ADR impact of inducing apoptosis of the tumor cell: ADR PND the effect of inducing apoptosis of the tumor cell in table 3. The joint application with the ADR PND, the K562/A02 a marked increase in the percentage of apoptosis. Table 3. PND joint application with the ADR to induce apoptosis Embodiment 1 takes the naphthyridine 0.1g g, by adding starch 1g, dextrin 0.5g, 10% dextrin 0.5g and stearic acid 0.005g, and the like, prepared into tablets. Embodiment 2 takes the naphthyridine 80 mg, physiological saline is added to 2 ml, the ampule encapsulates, circulation of steam for the 100 [...] 30 minutes for sterilization. Made of 80 mg/2 ml injection. The present invention discloses the application of PND in preparing antitumor medicines and tumor-reversing medicines with drug tolerance. 1, malaridine and its salt in the preparation of the application of the anti-tumor drugs. 2, malaridine and its salt in preparing reverse multi-drug resistance of tumor application of the medicament. SKOV3 9.73±0 . 08 0.62±0 . 09 K562 9.14±1 . 92 3.07±0 . 34 K562/A02 5.10±0 . 70 231.25±59 . 83 MCF-7 9.94±1 . 99 77.27±4 . 65 MCF-7/ADR 10.98±2 . 51 1.61±1 . 01 HL60 2.22±1 . 21 0.041±0 . 01 HL60/ADR 3.96 2.65±0 . 31 KB 18.66 4.51 KB/V200 11.64 193.91 A549 9.93±3 . 84 10.93±1 . 20 A549DDP 9.28±1 . 70 81.15±14 . 80 K562 3.07±0 . 34 1.82±0 . 08 1.86±0 . 09 2.56±0 . 04 2.41±0 . 39 2.69±0 . 11 ( 1.52±0 . 06) ( 1.47±0 . 07) ( 1.16±0 . 02) ( 1.16±0 . 20) ( 1.02±0 . 04) K562/A02 231.25±59 . 83 5.10±1 . 99 0.96 3.24±1 . 29 14.94±6 . 05 89.54±7 . 08 ( 49.05±19 . 12) ( 240.50) ( 79.75±31 . 48) ( 17.72±7 . 79) ( 2.60±0 . 22) MCF-7 1.61±1 . 01 1.23±0 . 03 0.86±0 . 46 1.21±0 . 07 1.36±0 . 35 1.47±2 . 58 ( 1.31±0 . 03) ( 2.16±1 . 14) ( 1.38±0 . 08) ( 1.23±0 . 34) ( 1.12±0 . 21) MCF-7/R 77.27±2 . 53 8.92±1 . 80 2.61±0 . 15 22.61±13 . 21 35.58±21 . 45 27.87±6 . 11 ( 8.84±1 . 78) ( 29.68±1 . 65) ( 4.30±2 . 38) ( 2.70±1 . 40) ( 2.84±0 . 62) PND (µm) 0 1.1 2.2 3.3 Apoptosis % 2.64 11.69 19.14 46.18