CN112675174B - New use of poly ADP ribose polymerase inhibitor for treating hepatitis B virus related diseases - Google Patents
New use of poly ADP ribose polymerase inhibitor for treating hepatitis B virus related diseases Download PDFInfo
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Abstract
The present invention relates to pharmaceutical compositions of poly (ADP ribose) polymerase (PARP) inhibitors, methods of using the same for treating or preventing hepatitis virus related diseases, and uses thereof. More particularly, the present invention relates to pharmaceutical compositions comprising (1- (cyclopropylformyl) -4- [ 5- [ (3, 4-dihydro-4-oxo-1-phthalazinyl) methyl ] -2-fluorobenzoyl ] piperazine), methods and uses thereof for treating or preventing diseases associated with hepatitis b virus infection, especially hepatitis b, liver cancer, and liver cirrhosis.
Description
Technical Field
The present invention relates generally to poly (ADP ribose) polymerase (PARP) inhibitors as therapeutic agents for treating or preventing hepatitis virus-related diseases. In particular, the present invention relates to phthalazinone compounds useful for treating or preventing diseases associated with hepatitis b virus infection, compositions comprising the same, methods of treatment, and uses thereof. More particularly, the invention relates to 1- (cyclopropane formyl) -4- [ 5- [ (3, 4-dihydro-4-oxo-1-phthalazinyl) methyl ] -2-fluorobenzoyl ] piperazine (also called Olaparib, Chinese Olaparib) for treating or preventing diseases related to hepatitis B virus infection, a pharmaceutical composition containing the inhibitor, a method for treating diseases related to hepatitis B virus (Hapatitis BVirus, hereinafter referred to as HBV) infection by using the inhibitor, and application of the inhibitor and the pharmaceutical composition.
Background
Acute hepatitis B virus infection is often converted into chronic infection, and chronic hepatitis B virus activity causes hepatocyte damage and necrosis, stimulates local immune response, and causes liver pathological changes including hepatic fibrosis, liver cirrhosis, liver cancer and the like.
At present, there is no standard treatment method which can stably ensure a certain degree of therapeutic effect on liver cancer, and liver cancer has high resistance to chemical drugs, and even if the goals of temporarily stabilizing tumors and prolonging life are to be achieved, it is difficult. And because the operation difficulty of intra-arterial injection is an important obstacle of clinical treatment, the clinical requirement for developing a new generation of liver cancer chemotherapy is particularly urgent, and the key is to discover a chemical drug capable of powerfully killing hepatitis B virus positive liver cancer cells.
Phthalazinone PARP enzyme inhibitors (including olaparib) are heterocyclic polyarylether derivatives which have the function of inhibiting the enzymatic activity of poly (ADP-ribose) polymerase (PARP) family. The poly (ADP-ribose) polymerase family currently has about 18 proteins.
The catalytic domains of these polymerase Proteins share a certain degree of homology, but their domains are largely different (Ame et al, BioEssays, 26(8): 882-. PARP in an activated state associated with DNA synthesizes poly ADP ribose chains on target nucleoproteins using NAD +. The phthalazinone PARP enzyme inhibitor olaparib can be used for treating or preventing breast/ovarian cancer (US2014/01315973a1, published 2014, 10 months and 23 days).
It has been previously reported that PARP inhibitors can be used to treat a variety of diseases associated with poly (ADP-ribose) polymerase (PARP), including neurodegenerative diseases (e.g., senile dementia, huntington's chorea, parkinson's disease); diabetes mellitus; diseases that are complicated by ischemia or ischemia-reperfusion, such as myocardial infarction and acute renal failure; circulatory diseases such as septic shock; and inflammatory diseases such as chronic rheumatism and the like (see, for example, CN102964354A, published: 3/13/2013). A series of patent applications of phthalazinone PARP inhibitors, including WO2002036576, WO2004080976 and WO200602, have been disclosed, and it is considered that phthalazinone and its derivatives may be used for treating the above metabolic, degenerative, inflammatory and tumor diseases, but until now, there has been no discovery that phthalazinone PARP inhibitors are applied to the treatment and/or prevention of hepatitis virus infection-related diseases.
Disclosure of Invention
The present invention is based, at least in part, on the inventors' first discovery that: PARP inhibitors phthalazinones, especially Olaparib (1- (cyclopropane formyl) -4- [ 5- [ (3, 4-dihydro-4-oxo-1-phthalazinyl) methyl ] -2-fluorobenzoyl ] piperazine, also called Olaparib), can obviously inhibit the growth and proliferation of HBV positive cells, for example, the PARP inhibitors can obviously inhibit the growth and proliferation of Hepatitis B Virus (HBV) whole genome transgenic human hepatocyte line HL 02-H1 and HBx (hepatitis B virus coding X gene) cells, and have no toxicity or killing effect in a pharmacological sense on hepatocytes which are not normally infected with the hepatitis B virus; in addition, the inventors have unexpectedly found that the PARP inhibitors phthalazinones, especially olaparib, can continuously and stably reduce serum HBV-DNA copy number and hepatitis b surface antigen (HBsAg) levels significantly. HBV positive liver cancer cells have the defect of gene damage repair, and the proliferation of the HBV positive liver cells can be effectively inhibited by using low-dose olaparib single medicine. The inventors also demonstrated in a mouse model that olaparib effectively inhibits and eliminates HBV positive liver cancer cells.
Olaparib is able to eliminate active hepatitis B virus infected cells in a short period of time without significant effect on uninfected HBV or quiescent cells, making it possible to specifically eliminate HBV virus-carrying cells. Thus, not only can the active replication of the hepatitis B virus be suppressed, but also the large-area liver injury can be avoided.
Accordingly, the present invention provides a new use of PARP inhibitors phthalazinones, in particular olaparib, for the treatment or prevention of hepatitis viruses, in particular hepatitis b virus-related liver diseases.
The inventor particularly finds that the liver cancer cells positive to the hepatitis B virus are specifically and highly sensitive to Olaparib, so that the Olaparib becomes a new-generation chemical drug for the liver cancer related to the hepatitis B virus.
Accordingly, in a first aspect, the present invention relates to the use of the PARP inhibitors phthalazinones, in particular olaparib, for the treatment and prevention of hepatobiliary diseases caused by hepatitis virus infections, including but not limited to hepatitis virus carriers, acute hepatitis, chronic hepatitis, liver cancer, cholangiocellular carcinoma caused by hepatitis virus infections.
In yet another aspect, the present invention relates to the use of the PARP inhibitors phthalazinones, particularly olaparib, for the treatment and prevention of hepatobiliary diseases caused by Hepatitis B Virus (HBV) infection, including but not limited to Hepatitis B Virus (HBV) carriers, acute hepatitis, chronic hepatitis, liver cancer, cholangiocellular carcinoma caused by HBV infection, physiological abnormalities (e.g., abnormal physiological indices, circulatory system symptoms, digestive system symptoms, etc.) caused by Hepatitis B Virus (HBV) infection.
In another aspect, the present invention provides novel potent chemical agents, phthalazinone PARP inhibitors, particularly 1- (cyclopropylformyl) -4- [ 5- [ (3, 4-dihydro-4-oxo-1-phthalazinyl) methyl ] -2-fluorobenzoyl ] piperazine, for killing Hepatitis B Virus (HBV) -bearing host cells, thereby eliminating HBV-infected cells in patients with hepatitis b virus. At the same drug concentration, the phthalazinone PARP inhibitor has no toxicity or killing effect on normal liver cells without HBV infection.
It is another object of the present invention to provide a method for treating or preventing chronic hepatitis b virus infection in a patient using olaparib, comprising control of hepatitis b virus infection titer and viral load, said method comprising administering to said patient an effective amount of olaparib.
It is a further object of the present invention to provide a method and use of olaparib for treating or preventing hepatitis viruses, in particular hepatitis b virus infectious liver fibrosis, cirrhosis of a patient, said method comprising administering to said patient an effective amount of olaparib.
It is a further object of the present invention to provide a method for treating or preventing advanced liver fibrosis, cirrhosis of a patient using olaparib, said method comprising administering to said patient an effective amount of olaparib.
The invention is also characterized in that the low-dose olaparib is applied to the treatment of hepatitis B virus positive tumors, including hepatitis B virus positive hepatocellular carcinoma/cholangiocarcinoma and the like.
The invention is characterized in that the combined medication of the Olaparib in the positive tumor treatment of the hepatitis B virus comprises the combined application of the Olaparib and radiotherapy and platinum anti-cancer drugs.
One of the objects of the present invention is to provide a composition comprising a poly (ADP-ribose) polymerase (PARP) inhibitor for use in the treatment or prevention of a hepatitis virus related disease.
In the scheme of the invention, the PARP inhibitor is a phthalazinone compound.
In the present invention, the phthalazinone compound is olaparib (1- (cyclopropylformyl) -4- [ 5- [ (3, 4-dihydro-4-oxo-1-phthalazinyl) methyl ] -2-fluorobenzoyl ] piperazine) or a pharmaceutically acceptable salt or ester thereof.
In the pharmaceutical composition of the present invention, it comprises a low unit dose of 1-45 mg of PARP inhibitor.
In the scheme of the invention, the PARP inhibitor phthalazinone compound is used for preparing a medicament for treating or preventing hepatitis B virus infection-related diseases.
In the embodiment of the present invention, the hepatitis b virus infection-related disease is acute hepatitis, chronic hepatitis, liver fibrosis, liver cirrhosis, liver cancer, cholangiocellular carcinoma, physiological abnormality (e.g. abnormal physiological index, circulatory system symptom, digestive system symptom, etc.) caused by hepatitis b virus infection.
In the scheme of the invention, the PARP inhibitor phthalazinone medicines are used for treating or preventing diseases related to hepatitis virus infection, more preferably hepatitis B virus infectious diseases, more preferably hepatitis B virus carriers, still more preferably hepatitis B virus infection, and most preferably acute hepatitis, chronic hepatitis, hepatic fibrosis, cirrhosis, liver cancer and cholangiocellular carcinoma related to hepatitis B virus infection.
In the embodiment of the present invention, the drug may be administered by oral or parenteral routes.
In the scheme of the invention, the medicament is combined with other medicaments for treating diseases related to hepatitis virus infection.
In the embodiment of the present invention, the other drug is selected from anticancer drugs, preferably the anticancer drug is a platinum, camptothecin or HDAC (histone deacetylase) inhibitor.
The present invention also provides a method for treating or preventing diseases associated with hepatitis virus infectivity, which comprises administering an effective amount of a composition comprising the PARP inhibitor phthalazinones.
In the present invention, hepatitis virus infection includes, but is not limited to, Hepatitis B Virus (HBV) infection.
In the present invention, the diseases associated with hepatitis virus infection refer to various diseases caused by said hepatitis virus infection, including but not limited to hepatitis virus carriers, acute and chronic infections, liver fibrosis, liver cirrhosis, liver cancer, etc.
In the present invention, the disease associated with hepatitis virus infection is a hepatitis b virus infectious disease, more preferably a hepatitis b virus carrier, still more preferably a hepatitis b virus infection, and most preferably acute hepatitis, chronic hepatitis, hepatic fibrosis, cirrhosis, liver cancer, cholangiocellular carcinoma associated with hepatitis b virus infection.
In the invention, the method also comprises the further administration of other hepatitis virus infectivity-related disease drugs, preferably platinum, camptothecin or HDAC inhibitors.
The olaparib has obvious treatment and prevention effects in HBV positive liver cells, liver cirrhosis, liver fibrosis, liver cancer and other models of human-nude mice.
Further, Olaparib is administered as a single agent in combination with radiation therapy, platins, camptothecins, HDAC inhibitors, or a combination thereof, directed against the proliferation of HBV positive transgenic hepatocytes. Olaparib single drug and combined drug such as radiotherapy, platinum, camptothecin, HDAC inhibitor and the like are carried out in a human-naked mouse model, and the proliferation of hepatitis B virus positive liver cells in the model is detected to achieve remarkable inhibition.
Olaparib is administered in combination with radiation therapy, platins, camptothecins, HDAC inhibitors, or combinations thereof, directed against the proliferation of HBV positive hepatocytes. The survival rate of the olaparib single drug can be lower than 1 per thousand at the final concentration of 1.5 MuM.
Olaparib single drug and combination with radiotherapy, platinums, or a combination thereof are significantly effective in human-nude mouse models of HBV positive hepatocytes, liver cancer.
Those skilled in the art recognize that the class of drugs that can be administered in combination with olaparib includes various drugs that interfere with DNA metabolism, replication, and repair, including platins (but not limited to cisplatin and carboplatin), camptothecins, doxorubicin, anthracycline antineoplastics, and the like.
In the present application, a low unit dose of a PARP inhibitor phthalazinone (e.g. olaparib) refers to a low unit dose of 1-40 mg (the dosage of olaparib single drug involved in an animal experiment of the present invention is 639mg per day after mouse-human dose conversion according to a standard pharmaceutical weight animal dose conversion factor, which is recommended to be taken approximately 20% less than 800mg per day in the instruction manual for the use of olaparib drugs; the minimum effective dosage of olaparib in a two-drug animal model experiment is 156.5mg after conversion, which is recommended to be taken more than 80% less than 800mg per day in the instruction manual for the use of olaparib drugs; subsequent animal experiments in progress of the present invention further suggest the use of lower doses of olaparib, and therefore there is a good reason to apply a lower dosage pharmaceutical specification than the existing 50mg specification capsule in the market), preferably 1-40 mg, more preferably 5-35 mg, more preferably 10-30, still more preferably 15-20 mg in the present invention, a low unit dose of 1-40 mg of the PARP inhibitor includes any single dose within the stated range, e.g. 1mg and 40mg unit doses.
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FIG. 1 shows that Olaparib induces chromosome breakage of hepatitis B virus whole genome transgenic human hepatocyte line HL 02-H1 cells and HBx (hepatitis B virus coding X gene) hepatocytes, but has no obvious influence on non-hepatitis B positive human hepatocyte cell line HL-7702 cells. The upper panel shows metaphase chromosomes prepared 24 hours after Olaparib treatment of cells with HL 02-H1 and human liver cell line HL-7702. Arrows indicate distinct star chromosomes and distorted chromosomes. The following figure is a statistical result of the above experiment, showing that olaparib significantly increases the number of star chromosomes occurring in HL 02-H1 and HL-7702-HBx cells.
FIG. 2 pharmacological experiment of Olaparib on HL 02-H1 cell model. HBV significantly increased the sensitivity of HL 02-H1 cells to Olaparib. The killing efficiency of Olaparib on HBV positive cells (HL 02-H1) is about 500 times higher than that of HL-7702 cells. The Olaparib shows high synergistic effect when combined with cisplatin and carboplatin as conventional chemotherapeutic drugs.
FIG. 3 shows that the influence of Olaparib on the hepatitis B positive liver cancer cell line HepG2.2.15 is detected by an MTT method, and the Olaparib can obviously inhibit the growth of HepG2.2.15.
FIG. 4 shows that both Olaparib single drug and platinum drug combination can significantly inhibit the growth of transplanted tumor formed by hepatitis B virus whole genome transgenic human hepatocyte line HL 02-H1 in human athymic nude mouse tumor model.
Figure 5. Olaparib was used to effectively reduce serum virus levels in a mouse model of hepatitis B virus and to maintain low levels of virus levels below the positive threshold 20 days after Olaparib withdrawal.
Figure 6. Olaparib was used to effectively reduce serum viral antigen levels in a mouse model of hepatitis B virus, and virus levels remained low below the positive threshold for 20 days after Olaparib dosing.
Detailed Description
The following non-limiting examples serve to illustrate selected embodiments of the present invention. It should be understood that variations in the proportions of the components shown and alternative elements will be apparent to those skilled in the art and are therefore within the scope of the embodiments of the invention
In the following examples, HL-7702 cell lines (human hepatocytes, also known as L-02, China academy of sciences type culture Collection cell Bank, accession No.: GNHu6), HepG2 (human hepatoma cells, or HEPG2, ATCC accession No. HB-8065), HepG2.2.15 (stable cell line obtained by transfecting HBV whole genome into recipient cell HepG 2), Nude mice (BALB/c-Nude, SPF grade, female, from Sichuan university laboratory animal center) were used for experiments in the present invention, but not for other purposes. Olaparib (KU0059436, Selleck), cisplatin (cispain, 131102, Supertrack Bio-pharmacological agent), carboplatin (carboplatin, H20020180, Zilu pharmaceuticals Co., Ltd.), hepatitis B virus surface antigen diagnostic kit (S10910113, Shanghai Kawawa bioengineering Co., Ltd.), and Hepatitis B Virus (HBV) nucleic acid amplification (PCR) fluorescent quantitative detection kit (S20030059, Haichouwa bioengineering Co., Ltd.) were used in examples. Various antibodies and various chemical reagents are commercially available products, and are used for experiments in the invention, but not for other purposes.
Example 1 Olaparib increases the probability of chromosome breakage in HL 02-H1 cells
1. Hepatitis B Virus (HBV) whole genome transgenic human liver cell line HL 02-H1
HL-7702 cells were cultured in 5cm × 5cm flasks to a density of 90%. After trypsinization, the cells were plated again in 6cm petri dishes for 3 plates in total and cultured overnight. 10 μ g of pcDNA3.1-HBV-wholeGenome plasmid was transferred to each plate of cells, and the transfection procedure was as follows: mu.g of plasmid and TurboFect transfection reagent (Thermo, # R0531) were added to Opti-MEM serum-free medium, mixed well and left to stand for 20 min. The mixture was added to overnight cultured cells and after 48 hours G418 was added for selection (final concentration 800. mu.g/ml). One month of continuous screening, surviving cells were digested, diluted to 4/ml with complete medium and the diluted cell culture was added to 96-well plates at 200 μ l per well to ensure that each well was monoclonal in the 96-well plate. Culturing for 2-3 weeks, collecting 100 μ l supernatant culture medium, determining hepatitis B surface antigen level with enzyme-linked reaction kit, and selecting the cell with the highest OD value. Digesting, putting into a cell bottle, amplifying, and obtaining the HL 02-H1 stable cell line. The HL 02-H1 cell line has been preserved in the China Center for Type Culture Collection (CCTCC for short, Wuhan city Wuchang Loojia mountain, zip code: 430072) at the 4 th and 3 th of 2015, with the preservation number: CCTCCC 201554.
Establishment of HL-7702-HBx cell line and its control cell line by lentivirus infection
HL-7702 cells were cultured in 5cm × 5cm flasks to a density of 90%. After trypsinization, the plates were re-plated to 3.5cm petri dishes and 12 plates were co-plated. The density was about 25%. The cells were cultured overnight in a 5% CO2 incubator at 37 ℃. The next day, infection was performed with control (pLV) lentiviruses carrying HBx gene and empty vector (HBx and empty plasmid control (PLV) lentiviruses were packaged according to the instructions in the Kjeldahl gene lentivirus packaging transfection kit), and HL-7702-pLV, HL-7702-HBx cell lines were established. Empty plasmid control (PLV) lentiviruses were packaged according to the instructions in the GeneCommen lentivirus packaging transfection kit, and the virus fluid was infected into HL-7702 cell line.
3. Specific cytotoxic effects of Olaparib on HBV positive cells HL 02-H1 and HL-7702-HBx.
Taking HL-7702, HL 02-H1, HL-7702-pLV and HL-7702-HBx cells, respectively inoculating the cells to 26 cm culture dishes, adding 1 mu M olaparib into one dish of each cell line when the cells are adhered to the wall, adding DMSO with the same volume into the other dish for dosing control, and treating overnight. Colchicine was added the following day at a final concentration of 200 ng/ml. Treatment for 1.5 hours, cell pellet was fixed according to the following procedure: trypsinized and washed 1 time with PBS. The waste solution was removed and 200. mu.l of the mixed cells remained. 6ml of 75mM KCl pre-heated at 37 ℃ was added, and the sample was gently shaken during the addition to avoid cell clumping. Incubate at 37 ℃ for 16-25 min. After incubation, 200. mu.l of a mixed fixative of methanol and glacial acetic acid (methanol: glacial acetic acid 3: 1) was added to the sample, mixed well, centrifuged at 1000rpm for 10 min. Removing supernatant, mixing the cells with the residual 50 μ l, adding methanol and glacial acetic acid stationary liquid 5ml, mixing, and standing at 4 deg.C for 20 min. Centrifuge at 1000rpm for 10min, leave a little fixative, and resuspend the cells. Repeat step 3-5 fixed cells 3 times. After the last centrifugation, 500. mu.l of the fixative was left and stored at 4 ℃ for one week. Soaking and washing the glass slide with alcohol for 2-3 times, adding a little double distilled water, and freezing at-20 deg.C for 10 min. Dropping the fixed cell sap from the position of more than 20cm onto a pretreated ice sheet, and drying the ice sheet in the air. The dried slides were stained with Giemsa stain for 10 min. And (4) flushing away excessive dyeing solution, drying the glass slide, and dropwise adding resin sealing sheets. The mounting pieces were photographed and counted by observing with an upright microscope at 100 times.
The experimental results are as follows: FIG. 1 shows the chromosomal abnormalities and statistics of HL-7702, HL 02-H1, HL-7702-pLV and HL-7702-HBx cells after DMSO and Olaparib treatment. The numbers of star chromosomes and chromosome aberrations in HL 02-H1 and HL-7702-HBx cells are much higher than those in the control groups HL-7702 and HL-7702-pLV, and the abnormality is caused by the fact that the cells tend to simply reconnect broken ends together when suffering from DNA breakage, the cells have extremely high cytotoxicity, and the cells can die due to large accumulation. The difference between the numbers of star chromosomes and chromosome aberrations in HL 02-H1 and HL-7702-HBx cells after adding Olaparib and the number of the star chromosomes in the control group is more obvious (statistical test of Tgao: p < 0.001). Therefore, the olaparib can obviously improve the generation quantity of the stellate chromosomes in HL 02-H1 and HBx cells, and therefore, the olaparib can be expected to have the effect of specifically and effectively killing HBV positive proliferating cells.
Example 2 Olaparib significantly inhibited the proliferation of hepatitis B virus whole genome transgenic human hepatoma cell line HL 02-H1 cells
To further confirm the inhibitory effect of olaparib on HBV-positive cell proliferation, the inhibitory effect of olaparib on HBx-positive malignant hepatocytes HL 02-H1 was tested using a clonogenic assay.
Cells HL-7702 as a control group and HBV positive HL 02-H1 cells were inoculated into a 10cm diameter culture dish, and drugs or DMSO of the same volume were added as a control after the cells adhered to the dish. 37 ℃ and 5% CO2After 10 days of culture, cells were fixed with methanol, stained according to giemsa kit instructions, and the number of clones was counted. The total number of the required pavers is determined according to the requirement of the concentration of the drug gradient. The final concentration of Olaparib is 0, 0.25, 0.5, 1, 1.5 μ M, the dose of cisplatin combined with Olaparib is 0.1, 0.05, 0.025, 0.001, 0.005 μ M, and the dose of carboplatin is 0.25, 0.5, 0.75, 1 μ M. Olaparib was 0.25. mu.M, and control group was 0. mu.M. Wherein the olaparib is continuously administered, and the cisplatin and the carboplatin are treated by 24-hour administration.
The counting results are shown in FIG. 2.
The experimental results are as follows: the data show that no matter single drug or double drugs are used in combination in the experiment, the olaparib has obvious inhibition effect on HL 02-H1 cells under low dose. In the experiment, the killing rate of Olaparib on HL 02-H1 cells reaches over 99 percent at the concentration of 1.5 mu M, and the cell survival rate is still over 20 percent when the dosage of the medicine for the same type of experiment of the hepatitis B virus negative cell line reaches 2mu M in the current literature report (J Biol Chem,2011,286:12157-12165, 2010); in the double-drug experiment, the concentration of the olaparib is 0.25 mu M, and the dosage is further reduced compared with the dosage used by a single drug; the dosage of the cisplatin is lower than the lowest dosage of the dosages used in the similar type cytotoxicity experiments reported in most literatures at presentConcentration (1-10. mu.M, Matrix Stiff modifications, chemical reaction and therapy in regenerative Cells Carcinoma Cells
Schrader, Timothy T Gordon-Walker hepatology.2011april; 53 (4); Aurora-A proteins chemististance in hepatocellulose carcinosa b y targeting NF-kappaB/microRNA-21/PTEN signaling pathway; kai Zhang, JingChen, Oncotarget.2014Dec; 12916-12935), namely 0.1 mu M, can inhibit HL 02-H1 cells by more than 99.9 percent. In most of the literatures, the dose of carboplatin is at least 5 μ M higher to produce significant inhibition (60-90% inhibition). In the experiment, the inhibition rate can reach 90% when the dose of the carboplatin reaches 0.5 mu M, and can reach more than 99% when the dose further reaches 1 mu M, which is very rare in all the cytotoxicity experiments related to the carboplatin at present, and shows that the carboplatin has high synergistic killing effect on the hepatitis B virus cell strain HL 02-H1 when being used together with the olaparib (the two are calculated as high synergistic effect according to the CI formula). The survival rate of HBV negative normal liver cells as a control group is within the range which is not influenced in the pharmacology sense under the same drug concentration in single-drug and double-drug experiments. According to our results, to obtain a HL 02-H1 cell inhibition rate of 99.8% or more, the concentration of the olaparib single drug is required: ICX, a — 1200nM, cisplatin single drug concentration: ICX, B600 nM; the concentration of Olaparib in the combined medicine is DA-250 nM, and the concentration of cisplatin in the combined medicine is DB-100 nM. The combined action index (CI) in the pharmacological experiment was calculated from the above data: CI0 is DA/ICX, A + DB/ICX, B is 250/1200+100/600 is 0.375,0.2 is less than or equal to CI0 is less than 0.4, thereby determining strong synergy between the two drugs.
Example 3 Experimental method for killing effect of Olaparib on hepatitis B virus positive liver cancer cell (HepG2.2.15)
Collecting liver cancer cells HepG2 and HepG2.2.15 at logarithmic phase, counting by using a cell counting plate, adjusting the concentration of cell suspension, adding the two cells into two 96-well plates respectively, adding 100ul of the two cells into each well, adjusting the density of the cells to 1000 cells per well, and filling the marginal wells with sterile PBS. 5% CO2, incubation at 37 deg.C until cell monolayer spreads to the bottom of the well, and adding gradient concentration Olaparib to make the final concentration of the drug in each well 0,2, 2.5, 3.5, 4.5. mu.M. Each hole is provided with 3 multiple holes. And setting a zero setting hole (culture medium, MTT and dimethyl sulfoxide). After incubation for 16-48 hours at 37 ℃ in 5% CO2, 20ul of MTT solution (5mg/ml, i.e.0.5% MTT) was added to each well and incubation was continued for 4 h. The culture was terminated and the culture medium in the wells was carefully aspirated. 150ul of dimethyl sulfoxide was added to each well, and the mixture was shaken on a shaker at a low speed for 10min to dissolve the crystals sufficiently. The absorbance of each well was measured at 490nm OD in an ELISA. And (4) reading by enzyme labeling, converting the OD value into the number of living cells according to the standard growth curve equation of HepG2 and HepG2.2.15 cells, and drawing a cell survival rate chart under the action of different concentrations of Olaparib.
The experimental results are as follows: the experimental results show that when the final concentration of Olaparib reaches above 2.5. mu.M, the survival rate of HepG2.2.15 cell line is obviously different from that of HepG2 (Tmp is less than 0.001), as shown in FIG. 3. This result demonstrates that olaparib has an extremely effective inhibitory effect against HBV positive hepatoma cells.
Example 4 Olaparib alone or in combination with platinum chemotherapy significantly inhibited the growth of transplants made by HL 02-H1 cells in nude mice
Randomly selecting 20 athymic female nude mice of 4-8 weeks of age, dividing into 4 groups, inoculating HL 02-H1 cells under the axilla and in the groin, each injection point is about 4x106And (4) one cell. The volume of the induced tumor is about 3-4 mm when HL 02-H1 is used3The injection of the drug is started. The drug dose is suitably reduced after reference to the clinical dose (determined after human-mouse conversion according to a pharmacological conversion factor), and the drug dose is as follows: the dosage of the Olaparib single medicine is 131.5 mg/kg/day. Olaparib 32 mg/kg/day and cisplatin (0.21mg/kg/4 day) in the experiment of drug combination with genotoxic chemotherapeutic drugs with DNA damage effects such as platinum class and the like. The DMSO volume in the control group was the same as in the olaparib group. Single-medicine experiment is carried out for 9 days continuously, and the body surface of the tumor is measured on 3, 6, 7, 8 and 9 days after the medicine is injectedLength, width, height; the two-drug experiment was performed by continuous injection for 14 days, and the length, width and height of the tumor were measured at 0, 7, 9, 12 and 14 days. According to an ellipsoid calculation formula: tumor volume size was calculated as length x width x height x 3.14/6. And calculating the inhibition ratio of each medicine group according to the tumor inhibition ratio (control group tumor volume-medicine group tumor volume)/control group tumor volume multiplied by 100 percent, and drawing a chart. Fig. 4 is the change in tumor volume for different drug treatments.
The experimental results are as follows: in the single medicine group, the HL 02-H1 induced tumor is inhibited all the time and even has a regressive phenomenon. In The double-drug experiment, The daily average dose of Cisplatin is about 1/5 (2 mg/kg/2 days for Cisplatin intraperitoneal injection mice, and daytime for administration, see Lishan mountain Ping et al, The effect of Maixuekang in combination with Cisplatin on The growth of subcutaneous transplanted tumor of MHCC97H human liver cancer nude mice in 2010, volume 28, phase 009 of Chinese medicine journal, or 4mg/kg/4 days, see The Role of line of Rich type Kidney Kinase Kinase 2(Pyk2) on Cisplatin Resistance in Hepatiolular poison Carcinoma, Wei g, Kevin T.P.Ng, Chris K.W.PLoS.2011; 6(11)), The Orlaparib is about 45% in low dose (according to ToC, The effective dose of Orlaparib in The equal human mice experimental model, 200mg/kg and 800 mg/kg/o for clinical dose, see To&Kim，The PARP inhibitors,veliparib and olaparib,are effective chemo preventive agents for delaying mammary tumor development in BRCA1-deficient mice，Cancer Prevention Research 7:698-707,2014；Kaufman&Shapira-Frommer, Olaparib monophagy in tissues with a d-advanced cancer and a germline BRCA1/2mutation. journal of clinical Oncology 33(3):244-250,2015), still can effectively inhibit HL 02-H1 induced tumors, and the variance analysis is carried out according to experimental data to determine that the two drugs have synergistic effect under the dosage of 1.64 mg/day of Olaparib and 0.015 mg/day of cisplatin.
Example 5 use of Olaparib effectively reduced serum viral levels in a mouse model of hepatitis B virus and inhibited viral level rebound after discontinuation of dosing
Experimental methods: the mice were randomly divided into 4 groups of 5 mice each. Each mouse was injected with HepG2.2.15 cells with HBV complete genome (4 spots, 4X10 in the underarm groin separately)6Individual cells). Then 100 μ l of tail vein blood is extracted every 4 days, and the HBV copy number in the blood is determined by using fluorescence quantitative PCR when the HBV-DNA copy number reaches 105The medication is started. The recommended dosage for human body is as follows: olaparib, 168 mg/kg/day; entecavir (entecavir), 0.07 mg/kg/day; lamivudine (lamivudine), 1.6 mg/kg/day. The control group was injected with the same volume of DMSO as olaparib. For 10 consecutive days, about 200. mu.l of tail vein blood was withdrawn each day, and the number of HBV copies in the blood was determined by quantitative PCR using fluorescence, and the drug was stopped on day 11. The day of initiation of administration was taken as 0 day. HBV copy number in blood was determined by fluorescence quantitative PCR at day 15, 20, 25, 28, 30 using 200. mu.l of each tail vein blood, and changes in HBV copy number in mouse blood were determined.
The experimental results are as follows: the copy number detection of HBV-DNA in serum is the most direct and reliable method for evaluating the effect of various HBV-related liver disease treating medicines. According to the guidance of lamivudine clinical application in 2000, the treatment of chronic hepatitis B virus infected patients in China aims to reduce serum HBVDNA, normalize alanine Aminotransferase (ALT), improve liver histological changes and reduce incidence of liver cirrhosis and liver cancer. As shown in FIG. 5, when the serum HBV-DNA content of the mouse model reached the clinically prescribed critical positive (HBV-DNA copy number: 10)5Time), the olaparib and the most common nucleoside liver disease drugs currently used clinically, namely entecavir (entecavir) and lamivudine (lamivudine), can control the serum HBV-DNA level within the clinical negative range within 10 days. However, within 20 days after drug withdrawal, the HBV-DNA level of the serum of the mice of the entecavir and lamivudine administration groups gradually increases back to the positive level; the serum HBV-DNA levels of Olaparib mice were always within a stable negative range. The serum HBV-DNA content in the DMSO control group is always above the positive critical value. This indicates that Olaparib overcomes all nucleosides currently used in hepatitis B virus infection-related diseases in the treatment of HBV-positive liver diseases of all kindsThe greatest weakness of antiviral clinical drugs: although the virus replication can be reduced, infected cells cannot be eliminated, and the infection level of the body is stably reduced.
Example 6 use of Olaparib effectively reduced serum viral antigen levels in a mouse model of hepatitis B virus and inhibited viral antigen rebound after discontinuation of dosing
The experimental method comprises the following steps: the mice were randomly divided into 4 groups of 5 mice each. HepG2.2.15 cells per mouse (4 spots, 4X10 injections in the underarm groin, respectively)6Individual cells). Then 100 mul tail vein blood is extracted every 4 days, and the HBV copy number in the blood is determined by utilizing fluorescence quantitative PCR when the HBV-DNA copy number reaches 105The medication is started. Blood sample of mice at 1-5, 15 and 20 days after administration is 200 mul, standing at room temperature for 2-3 hours, centrifuging at 3000rpm for 20min, collecting serum, and detecting HBV antigen HBsAg level according to the specification of the hepatitis B virus surface antigen diagnostic kit. The OD values obtained were plotted.
The experimental results are as follows: fig. 6 shows that after drug administration, olaparib and entecavir (entecavir) and lamivudine (lamivudine), which are the most commonly used nucleoside liver disease drugs in the clinic, can reduce the HBsAg level to the clinical HBsAg negative critical level within 5 days. Within 20 days after drug withdrawal, the HBsAg levels of the mice in the entecavir and lamivudine administration groups gradually increased back, but the HBsAg levels of the mice in the olaparib group were always within a stable negative range. This also suggests that unlike the short-term efficacy of entecavir and lamivudine, olaparib can clear infected cells and steadily reduce the level of body infection.
Claims (2)
1. Use of a Poly ADP Ribose Polymerase (PARP) inhibitor in the manufacture of a medicament for treating liver cancer or cirrhosis, the medicament comprising a PARP inhibitor and a chemotherapeutic agent, wherein the PARP inhibitor is (1- (cyclopropane formyl) -4- [ 5- [ (3, 4-dihydro-4-oxo-1-phthalazinyl) methyl ] -2-fluorobenzoyl ] piperazine), the chemotherapeutic agent is cisplatin, and the liver cancer or cirrhosis is liver cancer or cirrhosis caused by hepatitis B virus infection.
2. The use according to claim 1, wherein the unit dose of the phthalazinone compound is 1-40 mg.
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