TWI691333B - Use of CDK inhibitors and oncolytic viruses in preparing anti-tumor drugs - Google Patents

Abstract

The invention belongs to the field of biomedicine, and relates to the use of CDK (cyclin-dependent kinase, cyclin-dependent kinase) inhibitors and oncolytic viruses in the preparation of antitumor drugs. The present invention finds for the first time that CDK inhibitors can be used to prepare oncolytic virus antitumor synergists. The invention also relates to a pharmaceutical composition containing a CDK inhibitor and an oncolytic virus, a pharmaceutical kit containing a CDK inhibitor and an oncolytic virus, and a CDK inhibitor and an oncolytic virus in treating tumors, especially for the oncolytic virus Use in insensitive tumors.

Description

Use of CDK inhibitors and oncolytic viruses in preparing anti-tumor drugs

The invention belongs to the field of biomedicine, and relates to the use of the combination of CDK inhibitors and oncolytic viruses in the preparation of anti-tumor drugs.

Oncolytic virus is a type of replicable virus that selectively infects and kills tumor cells without damaging normal cells. Oncolytic virotherapy is an innovative tumor target treatment strategy. It uses natural or genetically engineered viruses to selectively infect tumor cells and replicate in tumor cells to achieve targeted lysis and killing. The role of tumor cells, but no damage to normal cells.

The M1 virus (Alphavirus M1) belongs to the genus Alphavirus (Alphavirus), which has good application effects in the preparation of antitumor drugs. For example, the Chinese mainland invention patent application 201410425510.3 discloses that M1 virus can selectively cause tumor cell death without affecting normal cell survival, and it has very good application prospects in anti-tumor. However, different tumors have different sensitivities to the M1 virus. For some tumors, when the M1 virus is used alone, the oncolytic effect is not satisfactory. For example, as described in the Chinese mainland invention patent application 201410425510.3, when M1 is used as an anti-tumor drug, its effect on colorectal cancer, liver cancer, bladder cancer and breast cancer is not as obvious as pancreatic cancer, nasopharyngeal cancer, prostate cancer and melanoma; while glial Tumors, cervical cancer, and lung cancer are the second ones; gastric cancer is the least significant. Screening compounds that increase the therapeutic effect of oncolytic viruses is expected to increase the anti-tumor spectrum and anti-tumor strength of oncolytic viruses.

The inventors previously used chrysophanol and its derivatives as anticancer synergists of M1 virus. Experiments have found that after 50μM of chrysophanol and (multiple infection (MOI) = 0.001) M1 virus, the survival of tumor cells The rate dropped to 39.6%.

The use of drugs may have some negative effects on the organism. However, if the dosage of the drug used can be reduced to a very low level, these negative effects will be greatly reduced, and the impact on the organism will be greatly reduced. This is undoubtedly a good way to reduce toxic and side effects, and it is also a combination of drugs. Another benefit beyond the medicinal effect.

However, for the oncolytic virus synergistic drugs, especially alpha virus synergistic drugs, the current research is still very little, and most of them work at the experimental dose of μM level.

The object of the present invention is to provide an oncolytic virus antitumor synergist.

Another object of the present invention is to provide an oncolytic antitoxic and antitumor synergist that can play a synergistic effect at a low dose.

Another object of the present invention is to provide an oncolytic virus anticancer synergist based on a precise mechanism, which can selectively enhance the killing effect of oncolytic viruses on tumor cells without affecting normal cells.

Another object of the present invention is to provide a use of a CDK inhibitor in the preparation of an oncolytic virus antitumor synergist.

Another object of the present invention is to provide an anti-tumor pharmaceutical composition, which can make the oncolytic virus exert better anti-tumor effect.

Another object of the present invention is to provide a safe and effective oncolytic virus synergistic drug for tumors that are not sensitive to oncolytic viruses.

The invention achieves the above object through the following technical solution: The inventor discovered through research and screening that the CDK inhibitor unexpectedly can enhance the oncolytic effect of oncolytic viruses.

The CDK inhibitor is a substance that inhibits CDK activity, or a substance that degrades CDK, or a genetic tool to reduce the level of CDK.

The cell cycle is an important part of cell life activities. The study found that the occurrence and development of various malignant tumors are closely related to the disorder of the cell cycle regulatory mechanism, so the tumor is also considered to be a cell cycle disease. CDK (cyclin-dependent kinase, cyclin-dependent kinase) is an important class of serine/threonine kinases, responsible for regulating the orderly progression of the cell cycle, so CDK inhibitors can block the cell cycle, control cell proliferation and have resistance Tumor activity. In recent years, CDK inhibitors have become a hot research topic in current anti-cancer drugs.

The inventors can significantly enhance the oncolytic effect of oncolytic viruses by inhibiting CDK. The inventors used a compound SNS-032 that inhibits CDK activity to cooperate with oncolytic viruses, especially M1 virus, to act on tumor cells. The experimental results found that SNS-032 can cooperate with oncolytic viruses to enhance the anti-tumor effect.

The present invention finds for the first time that CDK inhibitors can be used as antitumor synergists/drug resistance reversal agents of oncolytic viruses.

The present invention provides the use of CDK inhibitors in the preparation of oncolytic virus antitumor synergists/drug resistance reversal agents.

Drug resistance reversal agent means that when some alpha viruses are used as anti-tumor drugs to treat tumors, there are some tumors that are not very sensitive to oncolytic viruses, or that these tumors are resistant to oncolytic viruses. The oncolytic virus can be used in combination with a CDK inhibitor (as a drug resistance reversal agent) to reverse the resistance of the tumor to the oncolytic virus.

The CDK inhibitor is selected from compounds; preferably, the CDK inhibitor includes, but is not limited to, selected from the following compounds or derivatives having CDK inhibition, or pharmaceutically acceptable salts, solvates, and Variants and isomers: SNS-032, Dinaciclib and other compounds that inhibit CDK activity. The method of obtaining the compound is optional but not limited to: chemical separation or synthesis by oneself or purchase from commercial sources.

In a preferred embodiment of the present invention, the CDK protein inhibitor is SNS-032, and its structural formula is shown in Formula 1:

In another preferred embodiment of the present invention, the CDK protein inhibitor is Dinaciclib, and its structural formula is shown in Formula 2:

The CDK inhibitors also include tools for inhibiting CDK gene expression, including but not limited to tools such as genes, gene silencing, gene editing or knockout.

As an alternative embodiment, the CDK inhibitor is selected from DNA, RNA, PNA or DNA-RNA-hybrid. They can be single-stranded or double-stranded.

CDK inhibitors can include small inhibitory nucleic acid molecules, such as short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), ribozymes, and small hairpin RNA (shRNA), all of which can reduce or Eliminate the expression of CDK protein.

These small inhibitory nucleic acid molecules may include the first and second strands, and the two hybridize to each other to form one or more double-stranded regions, each strand is about 18 to 28 nucleotides in length, about 18 to 23 nucleotides , Or 18, 19, 20, 21, 22 nucleotides. In addition, single strands may also contain regions that can hybridize to form double strands, for example in shRNA molecules.

These small inhibitory nucleic acid molecules may include modified nucleotides while maintaining this ability to attenuate or eliminate CDK protein expression. Modified nucleotides can be used to improve in vitro or in vivo properties, such as stability, activity, and/or bioavailability. These modified nucleotides may contain deoxynucleotides, 2'-methyl nucleotides, 2'-deoxy-2'-fluoronucleotides, 4'-trinucleotides, locked nucleic acid (LNA) nucleosides Acid and/or 2'-O-methoxyethyl nucleotide, etc. Small inhibitory nucleic acid molecules, such as short interfering RNA (siRNA), may also contain 5'- and/or 3'-cap structures to prevent degradation by exonucleases.

In some embodiments, a double-stranded nucleic acid composed of small inhibitory nucleic acid molecules contains blunt, or pendant nucleotides at both ends. Other nucleotides may include nucleotides that cause misalignment, bulging, cycling, or swinging base pairs. Small inhibitory nucleic acid molecules can be formulated for administration, for example, by liposome encapsulation, or incorporated into other carriers (such as biodegradable polymer hydrogels, or cyclodextrins).

Alternatively, the CDK inhibitor further includes one or more of antibodies, functional fragments of antibodies, peptides, and peptoids. Wherein, the antibodies may be monoclonal antibodies, multiple antibodies, multivalent antibodies, multispecific antibodies (for example: bispecific antibodies), and/or antibody fragments linked to the CDK. The antibody may be a chimeric antibody, a humanized antibody, a CDR grafted antibody, or a human antibody. The antibody fragment may be, for example, Fab, Fab', F(ab')2, Fv, Fd, single chain Fv (scFv), disulfide-bonded FV (sdFv), or VL, VH domain. The antibody may be in a conjugated form, for example, combined with a label, a detectable label, or a cytotoxic agent. The antibody may be isotype IgG (eg: IgG1, IgG2, IgG3, IgG4), IgA, IgM, IgE or IgD.

The oncolytic virus is selected from one or more of alpha virus, adenovirus, vaccinia virus, measles virus, vesicular stomatitis virus, and herpes simplex virus; wherein, the alpha virus is selected from M1 virus, Tower virus (Getah virus). As a preferred embodiment, the oncolytic virus is selected from M1 virus, Geta virus, or a combination thereof.

The oncolytic viruses (M1 virus, Geta virus, adenovirus, vaccinia virus, measles virus, vesicular stomatitis virus, and herpes simplex virus) described in the present invention may particularly refer to currently existing viruses, but also Exclude some viruses that may occur naturally or have mutations (natural mutations, mandatory mutations, or selective mutations), genetic modification, sequence addition or deletion, or partial replacement. The oncolytic viruses described herein include viruses that have undergone the above changes. Preferably, the above changes do not affect the oncolytic virus to exert the effects described in the present invention. The CDK inhibitor is a substance (such as a compound, or amino acid sequence, nucleotide sequence, etc.) or tool that can knock down or affect the expression of the CDK gene or reduce the amount or activity of the CDK protein. A person skilled in the art can modify, replace, or change its inhibitory compound or genetic tool, but as long as it exerts the above-mentioned CDK inhibition effect, it belongs to the CDK inhibitor of the present invention and belongs to the homogenous replacement of the above-mentioned substances, compounds or tools, etc. .

In some embodiments, the alpha virus is the M1 virus with deposit number CCTCC V201423 (deposited at the Chinese Type Culture Collection, deposit date July 17, 2014). As a virus most likely derived from the same strain, Genbank Accession No. EF011023 records the sequence of a strain of M1. As a virus with up to 97.8% homology with M1 virus (Wen et al. Virus Genes. 2007; 35(3):597-603), the two viruses have high identity, and M1 virus is also The literature is classified as Getah-like virus. It can be expected that the two have similar efficacy. A single alpha virus strain can also be administered. In other embodiments, multiple strains and/or types of alpha viruses can also be used.

The invention also provides a pharmaceutical composition for treating tumors, which comprises a CDK inhibitor and an oncolytic virus. The present invention also provides a pharmaceutical kit for treating tumors, which includes a CDK inhibitor or a derivative thereof or a combination thereof, and an oncolytic virus. The difference between the drug kit and the composition is that the CDK inhibitor is different from the oncolytic virus dosage form, but it is packaged separately (for example: pills, or capsules, or tablets or ampoules, containing the CDK inhibitor; additional pills, Or capsules, or tablets or ampoules, containing oncolytic viruses). In some embodiments, oncolytic viruses, CDK inhibitors, and combinations of oncolytic viruses and CDK inhibitors may also contain one or more adjuvants. The adjuvant refers to an ingredient that can assist the therapeutic effect of the drug in the composition of the drug. The drug kit can also contain individually packaged CDK inhibitors and individually packaged oncolytic viruses. The CDK inhibitor in the drug kit and the oncolytic virus can be administered simultaneously or in any sequential order, for example, the CDK inhibitor is administered before the oncolytic virus, or the CDK inhibitor is administered after the oncolytic virus, Or both. In various embodiments, the patient may be a mammal. In some embodiments, the mammal can be a human.

The CDK inhibitors include, but are not limited to, compounds such as SNS-032 (Formula 1) or Dinaciclib (Formula 2) that inhibit CDK protein activity. Or for CDK gene expression suppression tools, including but not limited to gene interference, gene silencing and gene editing or knockout tools. As a preferred embodiment of the present invention, the CDK inhibitor is SNS-032.

The oncolytic virus is selected from one or more of alpha virus, adenovirus, vaccinia virus, measles virus, vesicular stomatitis virus, and herpes simplex virus; wherein, the alpha virus is selected from M1 virus and Tower virus. As a preferred embodiment, the oncolytic virus is selected from at least one of M1 virus and Getavirus.

In the composition or drug kit, the ratio of SNS-032 or Dinaciclib and oncolytic virus is optionally: 0.005~200mg: 10 ³ ~10 ⁹ PFU; preferably 0.005~50mg: 10 ⁴ ~10 ⁹ PFU; further preferably 0.005~20 mg: 10 ⁵ ~10 ⁹ PFU.

The preferred dosage is: SNS-032 or Dinaciclib (Dinaciclib) in the range of 0.01mg/kg to 200mg/kg, while the oncolytic virus has a MOI of 10 ³ to 10 ⁹ (PFU/kg); preferably SNS-032 or Dinaciclib is used in the range of 0.05 mg/kg to 200 mg/kg, while the oncolytic virus uses a MOI of 10 ⁴ to 10 ⁹ (PFU/kg); more preferably SNS-032 or Dana Sydney (Dinaciclib) used in the range of 0.05mg / kg to 20mg / kg, while the use of oncolytic virus titer of MOI from ¹⁰⁵ to 10 ⁹ (PFU / kg).

In one embodiment, the oncolytic virus is selected from the group consisting of M1 virus, Gata virus, adenovirus, vaccinia virus, measles virus, vesicular stomatitis virus and herpes simplex virus. Preferably, the oncolytic virus is at least one of M1 virus and Gastrovirus.

In one embodiment, the tumor is a solid tumor or a hematoma. In one embodiment, the solid tumor is liver cancer, colorectal cancer, bladder cancer, breast cancer, cervical cancer, prostate cancer, glioma, melanoma, pancreatic cancer, nasopharyngeal cancer, lung cancer, or gastric cancer. In a preferred embodiment, the tumor is a tumor that is insensitive to oncolytic viruses. In a more preferred embodiment, the tumor is a tumor that is insensitive to M1 oncolytic virus.

As an optional embodiment, the SNS-032 or Dinaciclib provided by the present invention may be injections, tablets, capsules, patches, kits, and the like. As a preferred embodiment, the potentiating drug of the present invention is an injection; preferably, intravenous injection may be used.

As a further preferred embodiment of the present invention: The present invention finds that CDK inhibitors, especially SNS-032, can increase the antitumor effect of oncolytic viruses, so as to improve the therapeutic effectiveness of oncolytic viruses as antitumor drugs. Cytological experiments show that the combined application of M1 virus and SNS-032 can significantly cause tumor cell morphological lesions, thereby significantly enhancing the inhibitory effect on tumor cells.

We combined SNS-032 and M1 virus on human hepatocellular carcinoma Hep3B strain, and unexpectedly found that when the antiviral compound SNS-032 and M1 virus were used in combination, they significantly increased tumor cell morphology and significantly reduced tumor cell survival rate. For example, in one embodiment of the present invention, when M1 virus (MOI=0.001) is used to treat liver cancer cells alone, the tumor cell survival rate is 79.9%. When 50nM SNS-032 is used to treat liver cancer cells alone, the tumor cell survival rate is still As high as 90.0%, when 50nM SNS-032 was used in combination with the same MOI M1 virus, the tumor cell survival rate dropped to 47.6%. Compared with the anti-tumor effect of M1 virus alone, when SNS-032 is used in combination with M1, the oncolytic effect is significantly improved. It can be seen that the greatly improved oncolytic effect when SNS-032 and M1 are used together is due to the synergistic mechanism between SNS-032 and M1 virus, not simply through the anti-tumor mechanism of SNS-032.

The oncolytic virus synergist used in the present invention also has a feature that it can obtain a significant synergistic oncolytic virus effect at an extremely low dosage. For example, even at such low (for example, as low as 50nM) dosage, the killing rate of SNS-032 on cancer cells is almost negligible (the survival rate of liver cancer cells is still as high as 90.0%). However, when also used at such a low dose with alpha virus (MOI = 0.001), the tumor cell survival rate dropped significantly to 47.6%. The use of very low doses can greatly reduce other toxic and side effects that the drug itself may cause, which reduces the impact of the drug on the organism. This is very important for patients undergoing cancer treatment.

Figure 1 shows that the combined treatment of SNS-032 and M1 virus significantly reduces the survival rate of human hepatocellular carcinoma Hep3B.

The following embodiments further illustrate the present invention, but the embodiments of the present invention are not limited to the following examples. Any equivalent changes or modifications made according to the principles or concepts of the present invention should be regarded as the scope of protection of the present invention.

Unless otherwise specified, the materials and experimental methods used in the present invention are conventional materials and methods.

"Selected" in the manual is connected to the selected object, which can be understood as, for example: "X selected from: A, B, C, ..., E" or "X selected from: A, B, C , ... and one or more of E", etc., can be understood that X includes one of A, B, C, ... E, or any combination of the two , Or any combination of multiple. At this time, it is not excluded that X also includes some other types of substances.

In addition to the specific CDK inhibitors mentioned above, the inhibitors of the present invention can also be selected from CDK inhibitors known in the prior art, or substances found to have CDK inhibitory effects through subsequent research.

Example 1 SNS-032 and M1 virus combined treatment significantly reduces the survival rate of human hepatocellular carcinoma Hep3B

Materials: Human hepatocellular carcinoma Hep3B (purchased from ATCC), M1 virus (deposit number CCTCC V201423), high-glucose DMEM medium (purchased from Corning), automatic enzyme binding immunosorbent analysis reader.

Method: a) Cell inoculation, drug treatment: select logarithmic growth phase cells, DMEM complete culture medium (containing 10% fetal bovine serum, 1% double antibody) to make a cell suspension, 4×10 ³ per well Density was seeded in 96-well culture plates. After 12 hours, the cells were completely adhered. The experiment was divided into a control group, a SNS-032 group alone, an M1 infection group and a SNS-032/M1 combination group. The dose used is: the dose used is: M1 virus (MOI = 0.001) infected cells; SNS-032 is 50 nM.

MTT reacts with intracellular succinate dehydrogenase: when cultured to 48h, add 20μl (5mg/ml) of MTT to each well and continue to incubate for 4 hours. At this time, microscopic examination can be observed, and the granular blue purple formed in live cells Formazan crystals.

Dissolve the formazan particles: carefully remove the supernatant, add 100 μl/well of DMSO to dissolve the formed crystals, shake on a micro-oscillator for 5 min, and then detect the optical density (OD value) of each well on the enzyme-linked detector with a wavelength of 570 nm. Cell survival rate = OD value of drug treatment group / OD value of control group × 100%.

Results: As shown in Figure 1, M1 virus (MOI=0.001) alone had a small survival rate inhibition effect on tumor cells Hep3B, the tumor cell survival rate reached 79.9%, and the tumor cell survival rate of 50nM SNS-032 treatment group It is still as high as 90.0%, however, when the same 50nM SNS-032 is used in combination with M1 virus (MOI=0.001) (SNS-032+M1), the tumor cell survival rate drops to 47.3%.

Claims (10)

A pharmaceutical composition for treating tumors, comprising: a CDK inhibitor; and an oncolytic virus, wherein the oncolytic virus is selected from alpha virus, adenovirus, vaccinia virus, measles virus, vesicular stomatitis virus, and herpes simplex One or more of the rash virus. The composition of claim 1, wherein the CDK inhibitor is a substance that inhibits CDK activity, a substance that degrades CDK, a genetic tool that reduces CDK levels, or any combination thereof. The composition of claim 1, wherein the CDK inhibitor is selected from compounds. The composition according to claim 1, wherein the CDK inhibitor is selected from the following compounds or derivatives having CDK inhibitory effect, or pharmaceutically acceptable salts, solvates, tautomers, and isomerism Structure: SNS-032, Dinaciclib. The composition according to claim 1, wherein the CDK inhibitor is one or more selected from the group consisting of antibodies, functional fragments of antibodies, peptides, and peptoids. The composition of claim 1, wherein the CDK inhibitor is a tumor targeting CDK inhibitor. The composition according to claim 1, wherein the alpha virus line is selected from the M1 virus and the tower At least one of the viruses. The composition of any one of claims 1-7, wherein the tumor is a solid tumor or a hematoma. The composition according to claim 8, wherein the solid tumor is liver cancer, colorectal cancer, bladder cancer, breast cancer, cervical cancer, prostate cancer, glioma, melanoma, pancreatic cancer, nasopharyngeal cancer, lung cancer or gastric cancer . The composition of claims 1-7, wherein the tumor is a tumor that is insensitive to oncolytic viruses.

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