WO2017036284A1

WO2017036284A1 - Combined application of direct or indirect agonist of epac and oncolytic virus

Info

Publication number: WO2017036284A1
Application number: PCT/CN2016/094314
Authority: WO
Inventors: 颜光美; 李凯; 肖晓; 胡骏; 梁剑开; 林园; 张海鹏; 邱鹏新; 朱文博; 银巍; 林穗珍
Original assignee: 广州威溶特医药科技有限公司
Priority date: 2015-09-06
Filing date: 2016-08-10
Publication date: 2017-03-09
Also published as: CN105233285A; CN105233285B

Abstract

Disclosed are a pharmaceutical composition containing a direct or indirect agonist of Epac and an oncolytic virus, medication set, and the use thereof in the preparation of medication for treating tumors, in particular, tumors insensitive to the oncolytic virus. Also disclosed is a method of producing an oncolytic virus in-vitro.

Description

Combination of Epac direct or indirect agonist with oncolytic virus

Technical field

The present invention relates to the use of a combination of an Epac direct or indirect agonist and an oncolytic virus for the treatment of a tumor, in particular to a pharmaceutical composition comprising an Epac direct or indirect agonist and an oncolytic virus, comprising a separately packaged Epac a direct or indirect agonist and a separately packaged oncolytic virus kit, a method for producing oncolytic virus in vitro, and a combination of an Epac or indirect agonist and an oncolytic virus for treating a tumor, particularly for the oncolytic virus Use in sensitive tumors.

Background technique

Tumors are derived from the accumulation of genetic and epigenetic changes in normal cells that drive normal cells into malignant tumors. This complex pathological process determines the diversity of mechanisms involved in the development, maintenance, and metastasis of different tumors. At present, surgical resection, chemotherapy and radiotherapy are commonly used methods for clinical treatment of tumors. However, surgical resection of tumors is prone to recurrence, and side effects of radiotherapy and chemotherapy are large. 15 to 20% of human cancers are associated with viral infections such as hepatitis B virus (HBV), hepatitis C virus (HCV) and liver cancer, human papillomavirus (HPV) and cervical cancer.

Oncolytic virus is a type of replicable virus that targets infections and kills tumor cells without destroying normal cells. Oncolytic virotherapy is an innovative tumor-targeted therapeutic strategy that selectively infects tumor cells with natural or genetically engineered viruses and replicates them in tumor cells for targeted lysis. It kills tumor cells but is harmless to normal cells.

Now oncolytic virus treatment mainly faces the following two problems. First, the anti-tumor spectrum of oncolytic viruses is relatively narrow: tumor cells that are sensitive to oncolytic viruses are accompanied by more viral replication; tumor cells that are insensitive to oncolytic viruses are accompanied by less viral replication. Second, in the body, over time, the replication of the virus is limited and slowly cleared by the body. Therefore, how to make tumor cells selectively and effectively increase the replication of oncolytic viruses is an urgent problem to be solved.

Summary of the invention

In one aspect, the invention provides a pharmaceutical composition for treating a tumor comprising an Epac direct or indirect agonist and an oncolytic virus. Another aspect of the invention provides a pharmaceutical kit for treating a tumor comprising an individually packaged Epac direct or indirect agonist and a separately packaged oncolytic virus.

In one embodiment, the Epac direct or indirect agonist is selected from the group consisting of cAMP analogs, adenosine A combination of one or more of an acid cyclase agonist and a phosphodiesterase inhibitor. The cAMP analog is selected from the group consisting of dibutyryl cyclic adenosine monophosphate (db-cAMP), 8-(4-thiochlorophenyl)-3', 5'-cyclic adenosine monophosphate (8-CPT-cAMP), 3' , 5'-cyclic adenosine monophosphate (Rp-cAMPS), 2'-oxo-(2-aminoethylformyl)-3', 5'-cyclic adenosine monophosphate (Rp‐2'-AEC ‐cAMPS/Rp‐2′‐EDA‐cAMPS), 8-‐(6-hexanediamino)‐3′, 5′-cyclic adenosine monophosphate (Rp‐8‐AHA‐cAMPS), agarose gel Fixed 8-(6-hexanediamino)-3',5'-cyclic adenosine monophosphate (Rp‐8‐AHA‐cAMPS‐Agarose), 8-‐(2-ethyldiamino)‐3' , 5'-cyclic adenosine monophosphate (Rp-8-AEA-cAMPS), 8-(2-ethylenediamine)-2'-oxo-methyl-3', 5'-cyclic adenosine monophosphate (8‐AET‐2'‐O‐Me‐cAMP), 8-‐(6-hexanediamino)‐2′-oxo-methyl-3′,5′-cyclic adenosine monophosphate (8‐AHA‐ 2'-O-Me-cAMP), 8-benzylthio- 2'-oxo-methyl-3', 5'-cyclic adenosine monophosphate (Sp‐8‐BnT‐2'‐O‐Me ‐cAMPS/"S-223"), 8-benzylthio-3',5'-cyclic adenosine monophosphate (Sp‐8‐BnT‐cAMPS/”S‐ 220"), 1 -N-oxy-3', 5'-cyclic adenosine monophosphate (1 - NO-cAMP), 3', 5'-cyclic adenosine monophosphate (cAMP), N‐6‐(2‐ Aminoethyl)‐3′,5′-cyclic adenosine monophosphate (Sp‐6‐AE‐cAMPS), 8‐(2‐[DY‐547]‐aminoethylthio)‐3′, 5′‐ Cyclic adenosine monophosphate, 8-(6-hexanediamino)- 2-chloro-3',5'-cyclic adenosine monophosphate (8-AHA-2-Cl-cAMP) and 8-(6-hexanediamine) A combination of one or more of the 3', 5'-cyclic adenosine monophosphate (8-AHA-cAMP). The adenylate cyclase agonist is selected from forskolin. The phosphodiesterase inhibitor is selected from the group consisting of 3-isobutyl-1-methylxanthine (IBMX), Cilomilast, Roflumilast, Luteolin, and Dyphylline, Rolipram, Sildenafil Citrate, Tadalafil, Vardenafil HCl Trihydrate, Pimobendan, GSK256066, PF‐2545920, Apremilast (CC‐10004), Cilostazol, Milrinone, Avanafil One or more of Aminophylline, Dipyridamole, Doxofylline, and Deltarasin.

In one embodiment, the oncolytic virus is selected from the group consisting of an M1 virus, a Gata virus, a Sindbis virus, a Sendai virus, a Coxsackie virus, a herpes simplex virus, a parvovirus, an adenovirus, an adeno-associated virus, and polio. Virus, Newcastle disease virus, vesicular stomatitis virus, measles virus, reovirus, retrovirus, vaccinia virus, influenza virus and their engineered strains. . In some embodiments of the invention, the oncolytic virus is an M1 virus or an engineered strain thereof.

In one embodiment, the tumor is a solid tumor or a hematoma. In one embodiment, the tumor is selected from the group consisting of liver cancer, colorectal cancer, bladder cancer, breast cancer, cervical cancer, prostate cancer, glioma, black Oncology, pancreatic cancer, nasopharyngeal cancer, lung cancer, stomach cancer, ovarian cancer and leukemia. In some embodiments, the tumor is a tumor that is insensitive to oncolytic viruses. In one embodiment, the tumor is a tumor that is insensitive to Ml oncolytic virus.

Another aspect of the present invention provides a method for producing an oncolytic virus, comprising: culturing a cell; inoculating the oncolytic virus in the cultured cell; adding an Epac direct or indirect agonist to the cultured cell; collecting the cell culture solution, separating and purifying The oncolytic virus is obtained. In one embodiment, the oncolytic virus is selected from the group consisting of an M1 virus, a Gata virus, a Sindbis virus, a Sendai virus, a Coxsackie virus, a herpes simplex virus, a parvovirus, an adenovirus, an adeno-associated virus, and polio. Virus, Newcastle disease virus, vesicular stomatitis virus, measles virus, reovirus, retrovirus, vaccinia virus, influenza virus and their engineered strains. In one embodiment, the oncolytic virus is an M1 virus or an engineered strain thereof.

Another aspect of the invention provides a method of promoting replication of an oncolytic virus in a tumor cell, comprising: administering to the tumor cell a therapeutically effective amount before, after or simultaneously with administering the oncolytic virus to the tumor cell Epac direct or indirect agonist.

A further aspect of the invention provides a method of treating a subject having a tumor with an oncolytic virus comprising: administering to the subject a therapeutically effective amount of the oncolytic virus; before, after or simultaneously with step (a), A therapeutically effective amount of an Epac direct or indirect agonist is administered to the subject.

The present invention demonstrates that Epac direct or indirect agonists can be used to aid in the treatment of tumors by oncolytic viruses. Cytological experiments have demonstrated that Epac agonists cause increased oncolytic viral replication in a variety of tumor cells; animal experiments have demonstrated that Epac agonists specifically increase the replication of oncolytic viruses within tumors, thereby inhibiting tumor growth.

The present invention demonstrates that Epac agonists have a selective increase in oncovirus replication and are safe. Epac agonists can selectively increase tumor cell viral replication, but have no effect on viral replication in normal cells, indicating that Epac agonists have tumor cell selectivity; Epac agonists can be selectively injected via tail vein in tumor-bearing nude mice. increasing the amount of tumor tissue oncolytic virus, whereas in normal tissues is low viral load, both the amount of viral difference of about 10 ² to 10 ⁶ times, further clarified the tumor selectivity Epac agonist. Simultaneous administration of Epac agonists and oncolytic viruses (such as M1 virus) did not affect the body weight and mental state of nude mice, indicating that the combination of the two drugs is safe.

DRAWINGS

Figure 1 cAMP pathway activation significantly increases oncolytic virus protein expression;

a) cAMP pathway activation significantly increases oncolytic virus M1 structural and non-structural proteins in tumor cells expression;

b) cAMP pathway activation does not increase expression of oncolytic viral M1 structural and non-structural proteins in normal cells.

Figure 2 cAMP pathway activation significantly increases the oncolytic virus titer;

a) cAMP pathway activation increases the replication of oncolytic virus M1 in tumor cells;

b) cAMP pathway activation does not increase replication of oncolytic virus M1 in normal cells.

Figure 3 cAMP pathway activation significantly inhibits the expression of antiviral factors;

A-f) M1 infection induces expression of antiviral factors;

G-i) Epac agonist inhibits M1-induced expression of antiviral factors.

Figure 4 cAMP pathway activation significantly increases the cytopathic effect caused by oncolytic virus M1;

a) cAMP pathway activation increases the cytopathic effect caused by oncolytic virus M1 in tumor cells;

b) cAMP pathway activation does not increase the cytopathic effect caused by oncolytic virus M1 in normal cells.

Figure 5 cAMP pathway activation significantly increases the anti-tumor effect of oncolytic viruses;

a) cAMP pathway activation increases the oncolytic effect of oncolytic viruses in tumor cells;

b) cAMP pathway activation does not increase the oncolytic effect of oncolytic viruses in normal cells.

Figure 6. Other Epac agonists significantly increase the anti-tumor effect of oncolytic viruses;

a) Forskolin and 8-CPT-cAMP can also significantly inhibit the growth of tumor cells in combination with M1;

b) Forskolin and 8-CPT-cAMP can also increase the titer of tumor cells.

Figure 7. Increased oncolytic effect of cAMP pathway activation by Epac, not PKA;

a) PKA interference does not abolish the increased oncolytic effect of cAMP pathway activation;

b) Epac1 interference can abolish the increased oncolytic effect of cAMP pathway activation;

c) Epac1, but not PKA, mediates an increase in viral proteins.

Figure 8. Activation of cAMP pathway increases replication of oncolytic virus in vivo;

a) cAMP pathway activation increases tumor-specific replication of oncolytic viruses. QRT-PCR was used to detect the tissue distribution of HCT116 tumor-bearing mice after intravenous cAMP pathway activator/M1 virus. **, p < 0.01.

Figure 9 Epac agonist combined with oncolytic virus effectively inhibits tumor growth in tumor-bearing mice;

a) Epac agonist and the effect on tumor volume in HCT116 tumor-bearing mice after intravenous injection of the virus M1, M1 represents M1 virus treated group, the control group represents the group OptiPRO ^TM SFM solvent medium, of cAMP are shown in Table 8-CPT-cAMP treatment groups. cAMP+M1 represents a combined treatment group of 8-CPT-cAMP and M1 virus;

b) the effect of Epac agonist and M1 virus on the tumor volume of Hep3B tumor-bearing mice after intravenous injection,

c) the effect of Epac agonist and M1 virus on tumor volume in Capan-1 tumor-bearing mice after intravenous injection,

Tumor volume was expressed as mean ± standard deviation, statistical analysis of variance analysis by repeated measures; ** indicates statistical test p < 0.01; i.v. indicates tail vein injection.

Figure 10 Epac activation significantly increases the oncolytic virus titer in the tool cells;

Db-cAMP significantly increased replication of oncolytic virus M1 in different tool cells.

detailed description

As used herein, the term "composition" refers to a formulation suitable for administration to a prospective animal subject for therapeutic purposes, comprising at least one pharmaceutically active component, such as a compound. Optionally, the composition further comprises at least one pharmaceutically acceptable carrier or excipient.

The term "pharmaceutically acceptable" means that the substance does not have such characteristics, that is, taking into account the disease or condition to be treated and the respective route of administration, this property will prevent a rational and cautious medical practitioner from taking it to the patient. substance. For example, for injectables, such materials are generally required to be substantially sterile.

As used herein, the terms "therapeutically effective amount" and "effective amount" mean that the amount of the substance and substance is one to prevent, alleviate or ameliorate one or more symptoms of the disease or condition, and/or prolong the survival of the subject being treated. It is vaild.

I. Oncolytic virus

A large number of reports have shown that in vitro, many viruses replicate in a variety of tumor cells and cause tumor cell death, such as Sindbis virus, Sendai virus, Coxsackie virus, herpes simplex virus, parvovirus, adenovirus. , adeno-associated virus, poliovirus, Newcastle disease virus, vesicular stomatitis virus, measles virus, reovirus, retrovirus, vaccinia virus and influenza virus. In addition, these viruses have proven effective in treating cancer animal models.

Oncolytic virus therapy is an innovative therapeutic strategy compared to traditional therapy. The oncolytic virus therapy is based on the idea of selecting or designing a virus that selectively replicates in tumor cells and causes tumor cell death. In recent years, researchers have found that some oncolytic viruses use tumor-specific immunodeficiency to selectively kill tumor cells, including adenovirus, vaccinia virus, measles virus, vesicular stomatitis virus and herpes simplex virus. Preclinical studies of these oncolytic viruses are also encouraging and can cause different species Tumor degeneration.

In recent years, large-scale randomized clinical trials of oncolytic virus anticancer therapy have been underway, such as phase III clinical trials of attenuated HSV-1 virus encoding GM-CSF factor in patients with melanoma metastases; in the head and neck A phase II clinical trial of the combination of reovirus and paclitaxel and carboplatin in a population of patients with tumor recurrence; and a comparison of the efficacy of JX-594 and the efficacy of supportive therapy for patients with liver cancer who were not treated with Sorafenib Randomized clinical trials, etc. These clinical trials compared the synergy between oncolytic virus therapy and other traditional anticancer therapies. They also explored the immunomodulatory function of transgenic oncolytic viruses and the role of the body in generating anti-tumor immune responses for tumor therapy.

In the present invention, the oncolytic virus may be a natural or engineered virus. Natural viruses are, for example, M1 virus, Gaeta virus, Newcastle disease virus, and the like. The engineered virus, also referred to as "engineering strain" in the present invention, utilizes genetic engineering and the like to perform various directional operations and transformations on the viral gene, thereby directionally changing and controlling the behavior and function of the virus. The purpose of engineering natural oncolytic viruses includes, but is not limited to, attenuating toxicity, improving targeting, improving efficacy, increasing safety, and the like. Means of engineering engineering include, but are not limited to, genetic mutations (such as site-directed mutagenesis), knockouts, insertion of foreign fragments, deletion of redundant fragments, and the like. Most of the oncolytic viruses available are engineered viruses such as HSV-1 virus, adenovirus, vaccinia virus and the like.

The range of cancers that can be treated with oncolytic viruses for anti-cancer treatment includes solid tumors or hematomas. In the present invention, cancers which can be treated by oncolytic virus for anticancer treatment are selected from the group consisting of liver cancer, colorectal cancer, bladder cancer, breast cancer, cervical cancer, prostate cancer, glioma, melanoma, pancreatic cancer, nasopharyngeal carcinoma. , lung cancer, stomach cancer, ovarian cancer and leukemia. In some embodiments of the invention, the tumor is a tumor that is insensitive to oncolytic viruses.

II. Epac and its agonists

cAMP (cyclic adenosine monophosphate) is one of the important second messengers in the cell. ATP is catalyzed by adenylate cyclase (AC) and can be degraded by phosphodiesterase. By changing the intracellular cAMP concentration, a variety of intracellular signal transduction pathways can be regulated, thereby regulating protein activity, gene expression, and achieving physiological functions. Many molecules such as prostaglandins bind to G-protein coupled receptors on the cell surface, activate adenylate cyclase, catalyze the production of cAMP by adenosine triphosphate (ATP), and the resulting intracellular cyclic adenosine monophosphate can be degraded by phosphodiesterase. Deactivate the signal path. Downstream of cAMP is mainly composed of cAMP-dependent lazy protein kinase A (PKA), cAMP-dependent guanylate exchange protein (Epac) and cAMP-regulated ion channels. composition.

Epac is a guanylate exchange protein with a cAMP binding domain and a guanylate exchange domain that can be activated by intracellular second messenger cAMP, thereby activating downstream Rap proteins and exerting many different biological functions, such as control. Adherence, invasion and metastasis of tumor cells.

The present inventors have unexpectedly discovered that agonistic Epac can specifically significantly enhance the replication of oncolytic viruses in tumor cells, thereby allowing oncolytic viruses to cause tumor cell death with greater efficiency. The inventors have found that Epac direct or indirect agonists increase the replication of oncolytic viruses against a variety of tumor cells. Treatment of tumor cells with Epac direct or indirect agonists with oncolytic viruses for 24, 48 and 72 hours significantly increased the replication of oncolytic viruses in tumor cells in a time-dependent manner. Therefore, when an Epac agonist is used in combination with an oncolytic virus, the effect of tumor treatment can be significantly enhanced.

In the present invention, Epac agonists can be classified into Epac direct agonists and Epac indirect agonists according to their mechanism of action. In the present invention, an Epac direct agonist is a compound or a combination of compounds that directly acts on Epac itself to agonize its expression; an Epac indirect agonist refers to a compound or combination of compounds that activates Epac ligand to agonize Epac expression.

Thus, cAMP and its analogs are Epac direct agonists that bind directly to the cAMP binding domain of Epac, thereby agonizing the expression of Epac.

A cAMP analog refers to any compound capable of mimicking the action of cAMP in this signaling pathway, which in the present invention may be selected from a combination of one or more of the following cAMP analogs: dibutyryl cyclic adenosine monophosphate (db-cAMP) , 8‐(4-thiochlorophenyl)‐3′, 5′-cyclic adenosine monophosphate (8‐CPT‐cAMP), 3′, 5′-cyclic adenosine monophosphate (Rp‐cAMPS), 2'-oxo-(2-aminoethylformyl)-3',5'-cyclic adenosine monophosphate (Rp‐2'‐AEC‐cAMPS/Rp‐2′‐EDA‐cAMPS), 8‐( 6-hexyldiamine)-3',5'-cyclic adenosine monophosphate (Rp-8-AHA-cAMPS), agarose gel-fixed 8-(6-hexanediamino)-3', 5'-cyclic adenosine monophosphate (Rp‐8‐AHA‐cAMPS‐Agarose), 8‐(2‐ethylenediamine)‐3′, 5′-cyclic adenosine monophosphate (Rp‐8‐AEA) ‐cAMPS), 8‐(2‐Ethylenediamine)‐2′-oxo-methyl-3′, 5′-cyclic adenosine monophosphate (8‐AET‐2′‐O‐Me‐cAMP), 8 ‐(6-hexanediamino)‐2′‐oxo-methyl-3′,5′-cyclic adenosine monophosphate (8‐AHA‐2′‐O‐Me‐cAMP), 8-benzylthio- 2'-oxo-methyl-3',5'-cyclic sulfur Adenosine Adenosine (Sp‐8‐BnT‐2'‐O‐Me‐cAMPS/“S‐223”), 8-Benzylthio-3', 5'-cyclic adenosine monophosphate (Sp‐8‐ BnT-cAMPS/"S-220"), 1‐N-oxy-3', 5'-cyclic adenosine monophosphate (1‐NO‐cAMP), 3′, 5′-cyclic adenosine monophosphate (cAMP), N‐6‐(2‐aminoethyl)‐3′, 5′-cyclic adenosine monophosphate (Sp‐6‐AE‐cAMPS), 8‐(2‐[DY‐547]‐aminoethylthio)‐3′,5′-cyclic adenosine monophosphate, 8-‐(6-hexanediamino)‐2‐chloro‐3′, 5′‐ ring Adenosine phosphate (8-AHA‐2‐Cl‐cAMP) and 8-‐(6-hexanediamino)-3',5′-cyclic adenosine monophosphate (8‐AHA‐cAMP).

The indirect agonist of Epac is, for example, an agent which can increase the intracellular cAMP content, such as an agonist of adenylate cyclase or an inhibitor of phosphodiesterase, and the like.

An adenylate cyclase agonist can agonize adenylate cyclase, promoting the production of cAMP from ATP, which in the present invention can be selected from Forskolin.

The phosphodiesterase inhibitor inhibits the degradation of cAMP by phosphodiesterase, which may be selected from the group consisting of: 3-isobutyl-1-methylpyrimidine (IBMX), cilostol, Roflumilast, Luteolin, Dyphylline, Rolipram, Sildenafil Citrate, Tadalafil , Vardenafil HCl Trihydrate, Pimobendan, GSK256066, PF-255020, Apremilast (CC‐10004), Cilostazol One or more of Milrinone, Avanafil, Aminophylline, Dipyridamole, Doxofylline, and Deltarasin.

III. Pharmaceutical Compositions and Administration

In the present invention, "pharmaceutical composition" means a composition comprising a compound and an oncolytic virus, wherein the compound and the oncolytic virus are present in the composition in a mixed form. The composition will generally be used in the treatment of human subjects. However, they can also be used to treat similar or identical conditions in other animal subjects. As used herein, the terms "subject", "animal object" and like terms mean human and non-human vertebrate, such as mammals, such as non-human primates, competitive animals, and commercial animals, such as horses, cows, pigs, sheep, Rodents, and pets (such as dogs and cats).

Suitable dosage forms will depend, in part, on the route of administration or administration, for example, orally, transdermally, transmucosally, by inhalation or by injection (parenteral). Such a dosage form should enable the compound/oncolytic virus to reach the target cell. Other factors are well known in the art and include considerations such as toxicity and delay in the dosage form in which the compound or composition exerts its effects. Techniques and formulations are generally found in The Science and Practice of Pharmacy, 21 ^st edition, Lippincott, Williams and Wilkins, Philadelphia, PA, 2005 (hereby incorporated by reference).

A carrier or excipient can be used to produce the composition. The carrier or excipient can be selected to facilitate administration of the compound. Examples of carriers include calcium carbonate, calcium phosphate, various sugars such as lactose, glucose or Sucrose), or starch type, cellulose derivative, gelatin, vegetable oil, polyethylene glycol, and physiologically compatible solvent. Examples of physiologically compatible solvents include sterile water for injection (WFI), saline solution, and glucose.

The composition or components of the composition can be administered by different routes, including intravenous, intraperitoneal, subcutaneous, intramuscular, oral, transmucosal, rectal, transdermal or inhalation. In some embodiments, injections or lyophilized powder injections are preferred. For oral administration, for example, the compounds can be formulated into conventional oral dosage forms such as capsules, tablets, and liquid preparations such as syrups, elixirs, and concentrated drops.

For inhalants, the compositions of the invention or components thereof may be formulated as a dry powder or a suitable solution, suspension or aerosol. The powders and solutions can be formulated with suitable additives known in the art. For example, the powder may comprise a suitable powder base such as lactose or starch, and the solution may include propylene glycol, sterile water, ethanol, sodium chloride, and other additives such as acids, bases, and buffer salts. This solution or suspension can be administered by inhalation via a spray, pump, nebulizer or nebulizer or the like. .

Pharmaceutical preparations for oral use can be obtained, for example by combining the composition or a component thereof with a solid excipient, optionally grinding the resulting mixture, and, if desired, processing a mixture of particles, if desired, thereby Get tablets or dragees. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol or sorbitol; cellulose preparations such as corn starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl fiber , hydroxypropyl methylcellulose, sodium carboxymethylcellulose (CMC) and/or polyvinylpyrrolidone (PVP: povidone). If necessary, a disintegrating agent such as crosslinked polyvinylpyrrolidone, agar or alginic acid or a salt thereof such as sodium alginate may be added.

Alternatively, injection (parenteral administration) may be used, such as intramuscular, intravenous, intraperitoneal, and/or subcutaneous. For injection, the compositions of the invention or components thereof are formulated as sterile liquid solutions, preferably in physiologically compatible buffers or solutions, such as saline solution, Hank's solution or Ringer's solution. Additionally, the compositions or components thereof can be formulated in solid form and reconstituted or suspended just prior to use. It is also possible to produce lyophilized powder forms.

Administration can also be by transmucosal, topical or transdermal means. For transmucosal, topical or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art and include, for example, for transmucosal administration, bile salts and fusidic acid derivatives. Additionally, detergents can be used to promote penetration. Transmucosal administration can, for example, be by nasal spray or suppository (transrectal or vaginal).

The topical formulations of the present invention may preferably be formulated as oils, creams, lotions, ointments, and the like, by selection of suitable carriers known in the art. Emulsifiers, stabilizers, wetting agents and antioxidants, as well as Color or fragrance formulations can also be included. Topically applied creams are preferably formulated from a mixture of mineral oil, self-emulsifying beeswax and water. In addition, transdermal administration includes a transdermal patch or dressing such as a bandage containing the active ingredient and optionally one or more carriers or diluents known in the art. For administration in the form of a transdermal delivery system, the dosage administered during the dosage regimen will of course be sustained rather than intermittent.

Effective amounts of the various components can be determined by standard procedures to be administered, for example, the considerations compound IC _50, the biological half life of the compound, the age, size and weight of the object-related disorders. The importance of these and other factors is well known to those of ordinary skill in the art. In general, the dosage will be between about 0.01 mg/kg to 50 mg/kg, preferably between 0.1 mg/kg and 20 mg/kg, of the subject being treated. Multiple doses can be used. As an alternative mode of administration, the Epac agonists of the invention may be administered by intravenous or intratumoral injection. Intratumoral injection is administered daily from 5 mg/kg to 500 mg/kg; 5 mg/kg to 5 g/kg is administered daily by intravenous injection.

The compositions of the invention or components thereof may also be used in combination with other therapeutic agents that treat the same disease. Such combination includes administration of these compounds and oncolytic viruses and one or more other therapeutic agents at different times, or the simultaneous use of such compounds with oncolytic viruses and one or more other therapeutic agents. In some embodiments, the dosage of one or more compounds/oncolytic viruses of the invention or other therapeutic agents used in combination may be modified, for example, by methods known to those skilled in the art to reduce the relative to the compound used alone. Or the dose of the therapeutic agent.

It is to be understood that the use or combination includes use with other therapies, drugs, medical procedures, and the like, wherein the other therapies or procedures may be at a different time than the compositions of the present invention or components thereof (eg, in the short term) (such as a few hours, such as 1, 2, 3, 4-24 hours) or for a longer period of time (such as 1-2 days, 2-4 days, 4-7 days, 1-4 weeks) or in this The composition of the invention or a component thereof is administered at the same time. The combined use also includes use with a therapy or medical procedure (such as surgery) that is administered once or infrequently, with the composition of the invention or a component thereof in the other Administration in a short or longer period of time before or after the therapy or procedure. In some embodiments, the invention is used to deliver a composition of the invention or a component thereof and one or more other pharmaceutical therapeutics, which pass Delivery by the same or different routes of administration.

Combination administration of any route of administration includes delivery of a composition of the invention or a component thereof and one or more other pharmaceutical therapeutics together in any formulation by the same route of administration, including chemically linking the two compounds and Formulations that maintain their respective therapeutic activity upon administration. In one aspect, the other drug therapy can be co-administered with a composition of the invention or a component thereof. Combined use by co-administration a formulation of a co-formulation or chemically linked compound, or a compound of two or more separate formulations in a short period of time (eg, within one hour, within 2 hours, within 3 hours, up to 24 hours) They are administered in the same or different routes.

Co-administration of separate formulations includes co-administration of delivery via one device, such as the same inhalation device, the same syringe, etc., or administered by different devices in a short period of time relative to each other. A co-formulation of a compound of the invention and one or more additional pharmaceutical therapies delivered by the same route of administration comprises preparing the materials together such that they can be administered by a device, including combinations of different compounds in one formulation, or compounds They are modified such that they are chemically linked together but still retain their respective biological activities. Such chemically linked compounds can include a linker that separates the two active ingredients, which are substantially maintained in vivo or which may degrade in vivo.

IV. Medicine kit

In the present invention, "pharmaceutical kit" means a pharmaceutical pack comprising an individually packaged oncolytic virus drug and a separately packaged Epac direct or indirect agonist drug, wherein the oncolytic virus and the agonist are each present in a suitable dosage form, thereby facilitating The two are applied separately at regular intervals. In some embodiments, the "pharmaceutical kit" can also include instructions for use, an applicator.

"Independent packaging" as used in the present invention refers to a form of preparation that is prepared separately. For example, in one embodiment of the kit of the invention, the oncolytic virus is in the form of a single dose injection and the Epac agonist is in the form of a tablet. In another embodiment of the invention, the oncolytic virus is in the form of a lyophilized powder needle and the Epac agonist is in the form of an injection. One skilled in the art will recognize that depending on the respective properties of the oncolytic virus and the Epac agonist, the two can be separately formulated into other suitable dosage forms for co-packaging into the same pharmaceutical package to form the present invention. Medicine kit.

The individually packaged oncolytic virus drug and the individually packaged Epac direct or indirect agonist drug are each present in the drug kit of the present invention in a suitable dosage. The dose can be a single dose or a combination of multiple doses such that the final dose is administered to the subject in a therapeutically effective amount. Specific dosages can be determined by one of ordinary skill in the art based on routine experimentation and experience.

V. Method

In one aspect, the invention provides a method of promoting replication of an oncolytic virus in a tumor cell, comprising: administering a therapeutically effective amount to the tumor cell before, after or simultaneously with administration of the oncolytic virus to the tumor cell Epac direct or indirect agonist.

Another aspect of the invention provides a method of treating a subject having a tumor using an oncolytic virus, comprising: (a) administering to the subject a therapeutically effective amount of the oncolytic virus; and (b) in step (a) A therapeutically effective amount of an Epac direct or indirect agonist is administered to the subject before, after, or at the same time.

In one embodiment, a therapeutically effective amount of the oncolytic virus is first administered to a subject, and a therapeutically effective amount of an Epac direct or indirect agonist is administered to the subject. In another embodiment, a therapeutically effective amount of an Epac direct or indirect agonist is first administered to the subject, and a therapeutically effective amount of the oncolytic virus is administered to the subject. In another embodiment, a therapeutically effective amount of an Epac direct or indirect agonist and a therapeutically effective amount of the oncolytic virus are administered to the subject at the same time.

Administration of oncolytic viruses and agonists can be performed at short intervals (eg, several hours, such as 1, 2, 3, 4-24 hours) or at longer intervals (eg, 1-2 days, 2-4 days, 4- 7 days, 1-4 weeks).

Still another aspect of the present invention provides a method for producing an oncolytic virus, comprising: culturing a cell; inoculating the oncolytic virus in the cultured cell; adding an Epac direct or indirect agonist to the cultured cell; and collecting the cell culture solution, The oncolytic virus is obtained by isolation and purification. In some embodiments of the invention, the cell is selected from the group consisting of Vero cells, CHO cells, and HEK293 cells.

In the method, the oncolytic virus is selected from the group consisting of an M1 virus, a Gata virus, a Sindbis virus, a Sendai virus, a Coxsackie virus, a herpes simplex virus, a parvovirus, an adenovirus, an adeno-associated virus, and polio. Virus, Newcastle disease virus, vesicular stomatitis virus, measles virus, reovirus, retrovirus, vaccinia virus, influenza virus and their engineered strains.

In the method, the tumor is a solid tumor or a hematoma. The tumor is selected from the group consisting of liver cancer, colorectal cancer, bladder cancer, breast cancer, cervical cancer, prostate cancer, glioma, melanoma, pancreatic cancer, nasopharyngeal cancer, lung cancer, gastric cancer, ovarian cancer or leukemia. Preferably, the tumor is a tumor that is insensitive to oncolytic viruses.

In the method, the Epac direct or indirect agonist is selected from the group consisting of a combination of one or more of a cAMP analog, an adenylate cyclase agonist, and a phosphodiesterase inhibitor. The cAMP analog is selected from the group consisting of dibutyryl cyclic adenosine monophosphate (db-cAMP), 8-(4-thiochlorophenyl)-3', 5'-cyclic adenosine monophosphate (8-CPT-cAMP), 3' , 5'-cyclic adenosine monophosphate (Rp-cAMPS), 2'-oxo-(2-aminoethylformyl)-3', 5'-cyclic adenosine monophosphate (Rp‐2'-AEC ‐cAMPS/Rp‐2′‐EDA‐cAMPS), 8-‐(6-hexanediamino)‐3′, 5′-cyclic adenosine monophosphate (Rp‐8‐AHA‐cAMPS), agarose gel Fixed 8-(6-hexanediamino)-3',5'-cyclic adenosine monophosphate (Rp‐8‐AHA‐cAMPS‐Agarose), 8-‐(2-ethyldiamino)‐3' 5'-cyclic adenosine monophosphate (Rp‐8‐AEA‐cAMPS), 8‐(2‐Ethylenediamine)‐2′-oxo-methyl-3′,5′-cyclic adenosine monophosphate (8‐AET‐2′‐O‐ Me‐cAMP), 8‐(6-hexanediamino)‐2′-oxo-methyl-3′, 5′-cyclic adenosine monophosphate (8‐AHA‐2′‐O‐Me‐cAMP), 8-benzylthio- 2'-oxo-methyl-3',5'-cyclic adenosine monophosphate (Sp‐8‐BnT‐2'‐O‐Me‐cAMPS/“S‐223”), 8-benzylthio-3',5'-cyclic adenosine monophosphate (Sp-8-BnT-cAMPS/"S-220"), 1-N-oxy-3', 5'-cyclic phosphate Glycosides (1‐NO‐cAMP), 3′, 5′-cyclic adenosine monophosphate (cAMP), N‐6‐(2‐aminoethyl)‐3′, 5′-cyclic adenosine monophosphate (Sp‐ 6‐AE‐cAMPS), 8‐(2‐[DY‐547]‐aminoethylthio)‐3′, 5′-cyclic adenosine monophosphate, 8-‐(6-hexanediamino)‐2‐chloro‐ 3',5'-cyclic adenosine monophosphate (8-AHA‐2‐Cl‐cAMP) and 8-‐(6-hexanediamino)‐3′,5′-cyclic adenosine monophosphate (8‐AHA‐cAMP) . The adenylate cyclase agonist is selected from the group consisting of Forskolin. The phosphodiesterase inhibitor is selected from the group consisting of 3-isobutyl-1-methylxanthine (IBMX), Cilomilast, Roflumilast, Luteolin, and Dyphylline, Rolipram, Sildenafil Citrate, Tadalafil, Vardenafil HCl Trihydrate, Pimobendan, GSK256066, PF‐2545920, Apremilast (CC‐10004), Cilostazol, Milrinone, Avanafil One or more of Aminophylline, Dipyridamole, Doxofylline, and Deltarasin. Preferably, the administration is intravenous.

Example 1 Epac agonists significantly increase the replication of oncolytic viruses.

1) Epac agonists significantly increase the expression of oncolytic viral proteins.

material:

Human colorectal cell carcinoma HCT116, human pancreatic cell carcinoma Capan-1, human immortalized normal liver cell line L-02, M1 virus, high glucose DMEM medium, db-cAMP (Sigma, USA). Western bolt: total cell protein extract (

Mammalian Protein Extraction Reagent, Thermo), E1 antibody (home made), NS3 antibody (home made), GAPDH antibody (Bioworld USA);

method:

a) Cell culture: human colorectal cancer cell line HCT116, human pancreatic cancer cell line Capan-1, human immortalized normal liver cell line L-02, grown in 10% FBS, 100 U/ml penicillin and 0.1 Mg/ml streptomycin in DMEM complete medium or primary cell culture medium; all cell lines were placed in 5% CO ₂ , cultured in 37 ° C constant temperature closed incubator (relative humidity 95%), inverted microscope observation growing situation. The cells were passaged in about 2 to 3 days, and the cells in the logarithmic growth phase were used for formal experiments.

b) Detection of viral proteins: Select logarithmic growth phase cells, DMEM complete medium (containing 10% fetal bovine serum, 1% double antibody) to make cell suspension, cells inoculated at a density of 3.0 × 10 ⁵ / dish in 10mm culture Inside the dish. After treating the cells with db-cAMP (1 mM)/M1 virus (MOI=10) for 24 hours, the expression of viral proteins was detected by western blot.

result:

As shown in Figure 1a, db-cAMP treatment significantly increased expression of Ml viral protein in tumor cells. In the control group and the db-cAMP group alone, no viral protein was detected; the virus protein expression was observed in the M1 virus group alone, but was significantly increased in the db-cAMP group and the M1 group. M1 viral structural protein E1 and structural protein NS3 expression levels. In addition, combined treatment of db-cAMP and M1 in normal cells did not increase M1 viral protein expression. As shown in Fig. 1b, the human immortalized normal liver cell line L-02 cells were infected with the same titer of M1 virus, and no viral protein expression was found in either the M1 group alone or the db-cAMP group and the M1 group. The above results indicate that activation of the cAMP pathway can selectively promote M1 virus replication in tumor cells, thereby increasing the expression of oncolytic virus M1 protein.

2) Epac agonist selectively increases the titer of oncolytic virus M1

material:

Human colorectal cell carcinoma HCT116, human pancreatic cell carcinoma Capan-1, human immortalized normal liver cell line L-02, M1 virus, high glucose DMEM medium, db-cAMP (Sigma, USA), BHK-21 cell line. M1 virus titer determination (TCID50 method):

(1) BHK-21 cells were cultured, and cells in logarithmic growth phase were seeded, and inoculated into a 96-well plate, 100 μl of the cell suspension was added to each well to make the cell volume reach 2 to 3 × 10 ⁵ /ml.

(2) The M1 virus solution was diluted 10 times in a row, from 10 ^{-1 to} 10 ^-10 .

(3) The diluted virus was inoculated into a 96-well microplate, and each dilution was inoculated with a total of 8 wells in a row, and 20 μl was inoculated per well.

(4) Set normal cell control, normal cell control for two longitudinal rows. (20 μl growth solution + 100 μl cell suspension)

(5) Observe and record the results day by day, generally need to observe 5-7 days.

(6) Calculation of the results, according to the Reed-Muench method or the Karber method.

TCID50 calculation method

(1) Reed-Muench two methods

CPE: cytopathic effect

Distance ratio = (50% higher than 50% lesion rate) / (% above 50% lesion rate - percentage below 50% lesion rate) = (91.6-50) / (91.6-40) = 0.8

lgTCID50=distance ratio × difference between logarithm of dilution + logarithm of dilution above 50% lesion rate

=0.8×(-1)+(-3)=-3.8

TCID50=10 ^-3.8 /0.1ml

Meaning: Dilution of the virus by 10 ^3.8 times inoculation of 100 μl can cause 50% of the cells to develop lesions.

The virus titer is: 10 ^3.8 TCID50/0.1ml=6.3×104TCID50/ml

(2) Karber method

lgTCID50=L-d(s-0.5)

L: logarithm of the highest dilution (-1 in this case)

D: the difference between the logarithm of the dilution (in this case, -1)

S: sum of positive hole ratios

lgTCID50=-1-1×(3.375-0.5)=-3.875

TCID50=10 ^-3.875 /0.1ml

Meaning: The virus was diluted 10 ^3.875 times, and inoculation of 100 μl caused 50% of the cells to develop lesions.

The virus titer is: 10. ^3.875 TCID50/0.1ml=7.5×10 ⁴ TCID50/ml

method:

a) Inoculation of cells, administration: Select logarithmic growth phase cells, DMEM complete medium (containing 10% fetal bovine serum, 1% double antibody) to make cell suspension, with a density of 4 × 10 ³ /well per well Inoculate in a 96-well culture plate. After 12 hours, the cells were completely adherent. The experiment was divided into M1 alone group and db-cAMP/M1 combination group. Experimental group: M1 virus (MOI=0.1) infected cells; combined group: db-cAMP (500 μM) and M1 virus (MOI = 0.1) treated cells. Cell supernatants were collected at 0, 24, 48, and 72 hours.

b) Using the previously prepared BHK-21 cells, the titer of the virus in the supernatant was determined by gradient dilution according to the method of TCID50.

result:

As shown in Figure 2a, the M1 virus alone infection can be time-dependently increased in the human pancreatic cancer cell line Capan-1 and the human colorectal cancer cell line HCT116. However, in the human immortalized normal liver cell line L-02, the amount of virus cannot be increased in a time-dependent manner. At 24, 48, and 72 hours, db-cAMP treatment significantly increased the replication of oncolytic virus M1 in the human pancreatic cancer cell line Capan-1 and human colorectal cancer cell line HCT116, but did not significantly increase the oncolytic virus M1. Replication in human immortalized normal liver cell line L-02 (Fig. 2b). It is further illustrated that db-cAMP selectively increases the replication of oncolytic virus M1 in tumor cells.

3) Epac1 agonists significantly inhibit M1-induced antiviral expression.

material:

Human colorectal cell carcinoma HCT116, M1 virus, high glucose DMEM medium, db-cAMP (Sigma, USA). RNA extraction reagent Trizol, real-time PCR instrument.

method:

a) Cell culture: human colorectal cancer cell line HCT116, grown in DMEM complete medium containing 10% FBS, 100 U/ml penicillin and 0.1 mg/ml streptomycin; placed in 5% CO ₂ at 37 ° C The culture was passaged in a thermostatic closed incubator (relative humidity 95%), and the growth was observed under an inverted microscope. The cells were passaged in about 2 to 3 days, and the cells in the logarithmic growth phase were used for formal experiments.

b) Antiviral factor expression: Select logarithmic growth phase cells, DMEM complete medium (containing 10% fetal bovine serum, 1% double antibody) to make cell suspension, cells inoculated at a density of 3.0 × 10 ⁵ / dish at 10mm Inside the culture dish. Real-time quantitative RT-PCR was used to detect the mRNA expression of antiviral factors after treatment with db-cAMP (1 mM)/M1 virus (MOI=10) at the indicated time points in the cell map.

result:

As shown in Figures 3a-f, M1 infection in HCT116 colorectal cancer cell lines demonstrated time-dependent induction of expression of these antiviral factors, including interferon-α (IFNA), interferon beta (IFNB), interferon Regulatory factor-3 (IRF-3), interferon regulatory factor-7 (IRF-7), and interferon-inducible genes (ISGs) MDA5 and IFIT1. As shown in Figure 3g-i, Epac agonists db-cAMP and Forskolin were able to significantly inhibit M1-induced expression of these antiviral factors 12 hours after infection with M1 virus.

Example 2 Epac agonist and M1 virus selectively cause tumor cell death

1) Epac agonist and M1 virus selectively cause tumor cell lesions

material:

Human colorectal cell carcinoma HCT116, human immortalized normal liver cell line L-02, M1 virus, high glucose DMEM medium, inverted phase contrast microscope.

method:

a) Cell culture: human colorectal cell carcinoma HCT116, human immortalized normal liver cell line L-02, grown in DMEM complete medium containing 10% FBS, 100 U/ml penicillin and 0.1 mg/ml streptomycin; All cell lines were placed in 5% CO ₂ and cultured in a constant temperature closed incubator (relative humidity 95%) at 37 ° C. The growth was observed under an inverted microscope. The cells were passaged in about 2 to 3 days, and the cells in the logarithmic growth phase were used for formal experiments.

b) Observation under a cell microscope: Select the logarithmic growth phase cells, DMEM complete medium (containing 10% fetal bovine serum, 1% double antibody) to make a cell suspension, and inoculate the cells at a density of 2.5×10 ⁴ /well in 24 Hole in the culture plate. After 48 hours of infection with M1 virus (MOI = 1), changes in cell morphology were observed under an inverted phase contrast microscope.

Result:

As shown in Fig. 4a, the morphology of the cells was observed under a phase contrast microscope. HCT116 cells and Capan-1 cells were monolayer adherent growth, and the cells were closely arranged and the phenotype was consistent. Compared with the control group, the M1 treatment group and the db-cAMP treatment group alone, after the combination of db-cAMP and M1 virus (MOI=1) for 72 hours, the number of cells was significantly reduced, and the morphology of the cells changed significantly, and the cell body contracted. It is spherical, and the refractive index is obviously enhanced, showing a death lesion. As shown in Figure 4b, db-cAMP in combination with M1 virus treatment had no morphological effects on normal cells. Infected human immortalized normal liver cell line L-02 cells with the same concentration of db-cAMP and the same titer of M1 virus, no significant changes in cell number and morphology were observed. The above indicates that db-cAMP combined with M1 virus treatment selectively increases tumor cell death morphological changes significantly, but has no effect on normal cells.

2) Epac agonist combined with M1 virus to reduce cell survival rate

material:

Human colorectal cell carcinoma HCT116, human pancreatic cell carcinoma Capan-1, human immortalized normal liver cell line L-02, M1 virus, high glucose DMEM medium, inverted phase contrast microscope.

method:

a) Inoculation of cells, administration: Select logarithmic growth phase cells, DMEM complete medium (containing 10% fetal bovine serum, 1% double antibody) to make cell suspension, with a density of 4 × 10 ³ /well per well Inoculate in a 96-well culture plate. After 12 hours, the cells were fully adhered. The experiment was divided into control group, db-cAMP group alone, M1 infection group and db-cAMP/M1 combination group. The doses used were: M1 virus (MOI = 10) infected cells; db-cAMP with different dose gradients.

b) MTT and intracellular succinate dehydrogenase reaction: when cultured to 72h, add 20μl (5mg/ml) of MTT to each well and continue to incubate for 4 hours. At this time, microscopically formed granular particles can be observed in living cells. Blue-purple nails crystallize.

c) Dissolving the formazan granules: carefully aspirate the supernatant, add DMSO 100 μl/well to dissolve the crystals, shake on a micro-vibrator for 5 min, and then measure the optical density (OD value) of each well with a wavelength of 570 nm on an enzyme-linked detector. ). Each set of experiments was repeated 3 times. Cell viability = OD value of the drug-treated group / OD value of the control group × 100%.

result:

As shown in Fig. 5a, M1 virus treatment alone had a smaller survival inhibition effect on tumor cells HCT116 and Capan-1, and db-cAMP combined with M1 treatment significantly caused cell survival inhibition. As shown 5b, human immortalized normal cells L-02, the same db-cAMP combined with M1 treatment did not cause significant cell survival inhibition. The above results indicate that db-cAMP combined with M1 virus treatment selectively reduces tumor cell survival significantly, but has no effect on normal cells.

As shown in Figure 6a, in order to further verify the effect of Epac activation on the oncolytic effect of oncolytic virus M1, we continued to use 8-CPT-cAMP and Forskolin Epac agonists, and found that they can also significantly cause cells in combination with M1 virus treatment. Survival inhibition. As shown in Figure 6b, these two Epac agonists also significantly increased the replication of oncolytic virus M1.

Example 3 Epac agonist enhances the oncolysis effect of M1 by Epac1, not PKA

1) Interfering with PKA does not abolish the oncolytic effect of cAMP activation enhancement

material:

Human colorectal cell carcinoma HCT116, M1 virus, high glucose DMEM medium, MTT, siRNA, RNAiMAX, inverted phase contrast microscopy.

method:

a) Cell culture: human colorectal cell carcinoma HCT116, grown in DMEM complete medium containing 10% FBS, 100 U/ml penicillin and 0.1 mg/ml streptomycin; cells were placed in 5% CO ₂ at 37 ° C The cells were cultured in a closed incubator (relative humidity 95%), and the growth was observed under an inverted microscope. The cells were passaged in about 2 to 3 days, and the cells in the logarithmic growth phase were used for formal experiments.

b) RNA interference: select logarithmic growth phase cells, DMEM complete medium (containing 10% fetal bovine serum, 1% double antibody) to make cell suspension, cells inoculated at a density of 2.5 × 10 ⁴ /well in 24-well culture Inside the board. Replace with non-double antibody, DMEM medium containing 10% fetal bovine serum, transfected with RNAiMAX 50nm is siRNA. Scrambled RNA was used as a control group. After 24 hours of interference, the cells were replaced with DMEM complete medium and infected with M1 virus. After 72 hours, MTT assayed cell survival rate.

result:

As shown in Figure 7a, after HCT116 cells were transferred to scrambled siRNA, db-cAMP/M1 combination significantly inhibited cell survival compared with the control group and db-cAMP or M1 alone, which means db- cAMP significantly enhances the oncolysis effect of the M1 virus. When three different siRNA fragments were used to interfere with PKA, the combined treatment of db-cAMP/M1 could still inhibit the growth of tumor cells in HCT116 cells compared with control cells, M1 alone treatment group and db-cAMP alone treatment group. As shown As shown in 7c, the PKA interference fragment has better interference efficiency, but interferes with PKA but does not block the increased viral protein.

2) Interfering with Epac1 to abolish the oncolytic effect of cAMP activation enhancement

material:

method:

result:

As shown in Figure 7b, after HCT116 cells were transferred to scrambled siRNA, db-cAMP/M1 combination significantly inhibited cell viability compared with the control group and db-cAMP or M1 alone, ie db- cAMP significantly enhances the oncolysis effect of the M1 virus. When three different siRNA fragments were used to interfere with Epac1, the db-cAMP/M1 combination treatment group did not significantly inhibit cell survival in HCT116 cells. As shown in Figure 7c, the Epac1 interference fragment has better interference efficiency and interferes with Epac1 to block increased viral proteins.

Example 4 Epac agonist/M1 virus combined treatment effectively inhibited tumor growth

1) Epac agonist selectivity increases M1 virus replication in tumor tissues

material:

4 weeks old female BALB/c nude mice, human colorectal cancer cell line HCT116, RNA extraction reagent Trizol, tissue homogenizer, real-time fluorescence quantitative PCR

method:

5×10 ⁶ HCT116 cells were subcutaneously injected into the dorsal side of 4 week old nude mice. After 6 days, M1 virus (3 × 10 ⁷ PFU) or 8-CPT-cAMP (20 mg/kg) and M1 virus (3 × 10 ⁷ PFU) were injected into the tail vein of each mouse for two consecutive days. Nude mice were sacrificed on the third day after administration, and tissue samples (including tumor, heart, liver, spleen, lung, kidney, brain, muscle) were collected, and tissue RNA was extracted. Then, the amount of M1 virus was detected by QRT-PCR.

result:

As shown in Fig. 8, in the subcutaneous HCT116 tumor model of nude mice, the M1 virus had less M1 viral RNA in the tumor tissue after M1 virus treatment alone. Compared with the M1 virus-treated group alone, the number of M1 viral RNA in the tumor of 8-CPT-cAMP (20 mg/kg) combined with M1 virus (3×10 ⁷ PFU) was more than 1000 times, and M1 in other normal tissues. There was no significant increase in viral RNA. It is indicated that 8-CPT-cAMP can selectively increase the replication of M1 virus in tumor tissues, and M1 virus is enriched in tumor tissues.

2) Epac agonist/M1 virus combined treatment effectively inhibits tumor growth in tumor-bearing mice

material:

M1 virus, human hepatoma cell line Hep3B, human colorectal cancer cell line HCT116, human pancreatic cancer cell line Capan-1, 4 weeks old female BALB/c nude mouse

method:

a) Tumor-bearing mouse model establishment: 5×10 ⁶ Hep3B, HCT116 or Capan-1 cells were injected into the dorsal subcutaneous of 4 weeks old BALB/c nude mice.

b) Intravenous administration: When the tumor volume of Hep3B cells reached about 50 mm ³ , M1 virus (3 × 10 ⁷ PFU / time) / Epac agonist was intravenously administered. OptiPRO ^TM SFM medium injection as a solvent control group. The length and width of the tumor were measured every three days. The volume of the tumor was based on the formula: length × width ² /2.

result:

After subcutaneous tumor-bearing Hep3B, HCT116, Capan-1 (Fig. 9a, b, c) nude mice model was established on BALB/c nude mice, solvent control, M1 virus, 8-CPT-cAMP, etc. were administered intravenously multiple times. 8-CPT-cAMP was combined with M1 virus to observe the tumor volume changes in nude mice. Statistics showed that 8-CPT-cAMP combined with M1 virus treatment significantly inhibited tumor growth in Hep3B/HCT116/Capan-1 tumor-bearing mice compared with the control group or the drug-treated group alone, and the mental state was good. Indicates 8-CPT-cAMP The M1 virus treatment is more effective in inhibiting tumor growth than the M1 virus alone and the 8-CPT-cAMP cAMP group alone, and the combined use is safe.

Example 5 M1 virus preparation method

material:

Vero African green monkey kidney cells, Chinese hamster ovary cells CHO, human embryonic kidney cells HEK293, high glucose DMEM medium, OptiPRO ^TM SFM medium (1 ×), M1 virus, 100mm cell culture dish, centrifuge

method:

The logarithmic growth phase cells were selected, and DMEM complete medium (containing 10% fetal bovine serum, 1% double antibody) was used to prepare a cell suspension, and the cells were seeded in a 100 mm cell culture dish. When the cells to be fused degree reaches 80% ~ 90%, Vero cells into OptiPRO ^TM SFM medium. An additional 50 μl (MOI = 0.01) of M1 virus was added, or 500 μM db-cAMP was treated, and when the cells developed a large area lesion (about 36 hours), the cell supernatant was collected. Centrifuge at 2000-3000 RPM for 5 min, carefully aspirate the supernatant, mix and disassemble, and store in a refrigerator at -80 °C.

result:

As shown in Figure 10, in Vero, CHO, HEK293 cells, M1 virus infection alone caused more viral replication, and when treated with the Epac agonist db-cAMP, viral replication was significantly increased. This suggests that Epac agonists have an effect of increasing the production of oncolytic virus M1.

Claims

A pharmaceutical composition for treating a tumor comprising:

(a) Epac direct or indirect agonists, and

(b) Oncolytic virus.
The pharmaceutical composition according to claim 1, wherein the oncolytic virus is selected from the group consisting of an M1 virus, a Gata virus, a Sindbis virus, a Sendai virus, a Coxsackie virus, a herpes simplex virus, a parvovirus, an adenovirus, and an gland. Related viruses, poliovirus, Newcastle disease virus, vesicular stomatitis virus, measles virus, reovirus, retrovirus, vaccinia virus, influenza virus, and engineered strains thereof.
The pharmaceutical composition according to claim 1, wherein the Epac direct or indirect agonist is selected from a combination of one or more of a cAMP analog, an adenylate cyclase agonist, and a phosphodiesterase inhibitor. .
The pharmaceutical composition according to claim 3, wherein said cAMP analog is selected from the group consisting of dibutyryl cyclic adenosine monophosphate (db-cAMP), 8-(4-thiochlorophenyl)-3', 5'-cyclic phosphate Adenosine (8-CPT-cAMP), 3', 5'-cyclic adenosine monophosphate (Rp-cAMPS), 2'-oxo-(2-aminoformyl)-3', 5'-ring Adenosine monophosphate (Rp‐2'‐AEC‐cAMPS/Rp‐2′‐EDA‐cAMPS), 8-‐(6-hexanediamino)‐3′, 5′-cyclic adenosine monophosphate (Rp) ‐8‐AHA‐cAMPS), agarose gel-fixed 8-(6-hexanediamino)-3',5'-cyclic adenosine monophosphate (Rp‐8‐AHA‐cAMPS‐Agarose), 8 ‐(2‐Ethylenediamine)‐3′,5′-cyclic adenosine monophosphate (Rp‐8‐AEA‐cAMPS), 8‐(2‐ethylenediamine)‐2′‐oxo-A ‐3',5'-cyclic adenosine monophosphate (8-AET‐2'‐O‐Me‐cAMP), 8-‐(6-hexanediamino)‐2′-oxo-methyl-3′, 5'-cyclic adenosine monophosphate (8-AHA-2'-O-Me-cAMP), 8-benzylthio- 2'-oxo-methyl-3', 5'-cyclic adenosine monophosphate ( Sp‐8‐BnT‐2'‐O‐Me‐cAMPS/“S‐223”), 8-benzylthio-3',5 -cyclic adenosine monophosphate (Sp‐8‐BnT‐cAMPS/“S‐220”), 1‐N-oxy-3′, 5′-cyclic adenosine monophosphate (1‐NO‐cAMP), 3′ , 5'-cyclic adenosine monophosphate (cAMP), N‐6‐(2‐aminoethyl)‐3′, 5′-cyclic adenosine monophosphate (Sp‐6‐AE‐cAMPS), 8‐(2 ‐[DY‐547]‐aminoethylthio)‐3′,5′-cyclic adenosine monophosphate, 8-‐(6-hexanediamino)‐2‐chloro-3′,5′-cyclic adenosine monophosphate ( A combination of one or more of 8‐AHA‐2‐Cl‐cAMP) and 8-(6-hexanediamino)-3',5′-cyclic adenosine monophosphate (8‐AHA‐cAMP).
The pharmaceutical composition according to claim 3, wherein the adenylate cyclase agonist is forskolin.
The pharmaceutical composition according to claim 3, wherein the phosphodiesterase inhibitor is selected from the group consisting of 3-isobutyl-1-methylxanthine (IBMX), cilostol, and roflumilast. (Roflumilast), Luteolin, Dyphylline, Rolipram, Sildenafil Citrate, Tadalafil, Vardena Non-trihydrate hydrochloride (Vardenafil HCl Trihydrate), Pimobendan, GSK256066, PF-2552020, Apremilast (CC-10004), Cilostazol, Cyanamide One or more of melrinone, Avanafil, Aminophylline, Dipyridamole, Doxofylline, and Deltarasin.
The pharmaceutical composition according to claim 1, wherein the tumor is a solid tumor or a hematoma.
The pharmaceutical composition according to claim 1, wherein the tumor is selected from the group consisting of liver cancer, colorectal cancer, bladder cancer, breast cancer, cervical cancer, prostate cancer, glioma, melanoma, pancreatic cancer, nasopharyngeal cancer, lung cancer , stomach cancer, ovarian cancer and leukemia.
The pharmaceutical composition according to claim 1 or 2 or 3, wherein the tumor is a tumor that is insensitive to the oncolytic virus.
The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, and the pharmaceutical composition is in a form selected from the group consisting of lyophilized powder, injection, tablet, capsule, and drops. Or patch.
A pharmaceutical kit for treating tumors, comprising:

(a) individually packaged Epac direct or indirect agonists, and

(b) Individually packaged oncolytic viruses.
The pharmaceutical kit according to claim 11, wherein the oncolytic virus is selected from the group consisting of an M1 virus, a Gata virus, a Sindbis virus, a Sendai virus, a Coxsackie virus, a herpes simplex virus, a parvovirus, an adenovirus, and an adeno-associated virus. Virus, poliovirus, Newcastle disease virus, vesicular stomatitis virus, measles virus, reovirus, retrovirus, vaccinia virus, influenza virus and their engineered strains.
The pharmaceutical kit according to claim 11, wherein the Epac direct or indirect agonist is selected from the group consisting of a combination of one or more of a cAMP analog, an adenylate cyclase agonist, and a phosphodiesterase inhibitor.
The pharmaceutical kit according to claim 13, wherein said cAMP analog is selected from the group consisting of dibutyryl cyclic phosphate Adenosine (db-cAMP), 8-(4-thiochlorophenyl)-3', 5'-cyclic adenosine monophosphate (8-CPT-cAMP), 3', 5'-cyclic adenosine monophosphate (Rp‐cAMPS), 2'-oxo-(2-aminoformyl)-3',5'-cyclic adenosine monophosphate (Rp‐2'‐AEC‐cAMPS/Rp‐2'‐EDA‐ cAMPS), 8-(6-hexanediamino)-3',5'-cyclic adenosine monophosphate (Rp-8-AHA-cAMPS), agarose gel-fixed 8-(6-hexanediamine) )3',5'-cyclic adenosine monophosphate (Rp‐8‐AHA‐cAMPS‐Agarose), 8‐(2‐ethylenediamine)‐3′, 5′-cyclic adenosine monophosphate (Rp‐8‐AEA‐cAMPS), 8‐(2‐Ethylenediamine)‐2′-oxo-methyl-3′,5′-cyclic adenosine monophosphate (8‐AET‐2′‐O‐ Me‐cAMP), 8‐(6-hexanediamino)‐2′-oxo-methyl-3′, 5′-cyclic adenosine monophosphate (8‐AHA‐2′‐O‐Me‐cAMP), 8-benzylthio- 2'-oxo-methyl-3',5'-cyclic adenosine monophosphate (Sp‐8‐BnT‐2'‐O‐Me‐cAMPS/“S‐223”), 8-Benzylthio- 3',5'-cyclic adenosine monophosphate (Sp‐8‐BnT‐cAMPS/“S‐220”), 1‐N-oxy-3', 5′-cyclophosphate Acid adenosine (1‐NO‐cAMP), 3′, 5′-cyclic adenosine monophosphate (cAMP), N‐6‐(2‐aminoethyl)‐3′, 5′-cyclic adenosine monophosphate ( Sp‐6‐AE‐cAMPS), 8-‐(2‐[DY‐547]‐aminoethylthio)‐3′, 5′-cyclic adenosine monophosphate, 8-‐(6-hexanediamine)‐2‐ Chloro-3',5'-cyclic adenosine monophosphate (8-AHA‐2‐Cl‐cAMP) and 8-‐(6-hexanediamino)‐3′,5′-cyclic adenosine monophosphate (8‐AHA‐ A combination of one or more of cAMP).
The pharmaceutical kit according to claim 13, wherein the adenylate cyclase agonist is forskolin.
The pharmaceutical kit according to claim 13, wherein said phosphodiesterase inhibitor is selected from the group consisting of 3-isobutyl-1-methylxanthine (IBMX), cilostol, and roflumilast ( Roflumilast), Luteolin, Dyphylline, Rolipram, Sildenafil Citrate, Tadalafil, Vardenafil Vardenafil HCl Trihydrate, Pimobendan, GSK256066, PF-2552020, Apremilast (CC-10004), Cilostazol, Cetylpyridone One or more of (Milrinone), Avanafil, Aminophylline, Dipyridamole, Doxofylline, and Deltarasin.
The pharmaceutical kit according to claim 11, wherein the tumor is a solid tumor or a hematoma.
The pharmaceutical kit according to claim 11, wherein the tumor is selected from the group consisting of liver cancer, colorectal cancer, bladder cancer, breast cancer, cervical cancer, prostate cancer, glioma, melanoma, pancreatic cancer, nasopharyngeal cancer, lung cancer, Gastric cancer, ovarian cancer and leukemia.
The pharmaceutical kit according to claim 11 or 12 or 13, wherein said tumor is for said oncolytic disease Toxic insensitive tumors.
A method of producing an oncolytic virus, comprising:

(a) culturing the cells;

(b) inoculation of the oncolytic virus in the cultured cells;

(c) adding an Epac direct or indirect agonist to the cultured cells;

(d) collecting the cell culture solution, and isolating and purifying the oncolytic virus.
The method according to claim 20, wherein the oncolytic virus is selected from the group consisting of an M1 virus, a Gata virus, a Sindbis virus, a Sendai virus, a Coxsackie virus, a herpes simplex virus, a parvovirus, an adenovirus, and an adeno-associated virus. , poliovirus, Newcastle disease virus, vesicular stomatitis virus, measles virus, reovirus, retrovirus, vaccinia virus, influenza virus and their engineered strains.
The method of claim 20 wherein said cells are selected from the group consisting of Vero cells, CHO cells, and HEK293 cells.
The method of claim 20, wherein the Epac direct or indirect agonist is selected from the group consisting of a combination of one or more of a cAMP analog, an adenylate cyclase agonist, and a phosphodiesterase inhibitor.
The method according to claim 23, wherein said cAMP analog is selected from the group consisting of dibutyryl cyclic adenosine monophosphate (db-cAMP), 8-(4-thiochlorophenyl)-3', 5'-cyclic adenosine monophosphate ( 8‐CPT‐cAMP), 3′, 5′-cyclic adenosine monophosphate (Rp‐cAMPS), 2′-oxo-(2‐aminoformyl)‐3′, 5′-cyclic thiophosphate Adenosine (Rp‐2'‐AEC‐cAMPS/Rp‐2′‐EDA‐cAMPS), 8-‐(6-hexanediamino)‐3′, 5′-cyclic adenosine monophosphate (Rp‐8‐ AHA-cAMPS), agarose gel-fixed 8-(6-hexanediamino)-3',5'-cyclic adenosine monophosphate (Rp‐8‐AHA‐cAMPS‐Agarose), 8‐(2 -Ethylenediamine)-3',5'-cyclic adenosine monophosphate (Rp-8-AEA-cAMPS), 8-(2-ethanediamine)-2'-oxo-methyl-3 ',5'-cyclic adenosine monophosphate (8-AET‐2'‐O‐Me‐cAMP), 8-‐(6-hexanediamino)‐2′‐oxo-methyl-3′, 5′‐ Cyclic adenosine monophosphate (8-AHA‐2'‐O‐Me‐cAMP), 8-benzylthio- 2′-oxo-methyl- 3′, 5′-cyclic adenosine monophosphate (Sp‐8) ‐BnT‐2'‐O‐Me‐cAMPS/“S‐223”), 8-benzylthio- 3', 5'-cyclic sulfur Adenosine phosphate (Sp‐8‐BnT‐cAMPS/“S‐220”), 1‐N-oxy-3′, 5′-cyclic adenosine monophosphate (1‐NO‐cAMP), 3′, 5′‐ Cyclic adenosine monophosphate (cAMP), N‐6‐(2‐aminoethyl)‐3′, 5′-cyclic adenosine monophosphate (Sp‐6‐AE‐cAMPS), 8‐(2‐[DY‐ 547]-Aminoethylthio)-3',5'-cyclic adenosine monophosphate, 8-(6-hexanediamino)-2-chloro-3',5'-cyclic adenosine monophosphate (8-AHA- A combination of one or more of 2‐Cl‐cAMP) and 8-(6-hexanediamino)-3',5'-cyclic adenosine monophosphate (8-AHA-cAMP).
The method of claim 23, wherein the adenylate cyclase agonist is forskolin.
The method according to claim 23, wherein said phosphodiesterase inhibitor is selected from the group consisting of 3-isobutyl-1-methylxanthine (IBMX), Cilomilast, and Roflumilast. ), Luteolin, Dyphylline, Rolipram, Sildenafil Citrate, Tadalafil, Vardenafil Hydrate hydrochloride (Vardenafil HCl Trihydrate), Pimobendan, GSK256066, PF-2552020, Apremilast (CC-10004), Cilostazol, Cipropirone (Cilostazol) Milrinone), one or more of Avanafil, Aminophylline, Dipyridamole, Doxofylline, and Deltarasin.
The method of claim 20 wherein said cells are selected from the group consisting of Vero cells, CHO cells, and HEK293 cells.
Use of a combination of an Epac direct or indirect agonist and an oncolytic virus for the preparation of a medicament for treating a tumor.
The use according to claim 28, wherein the oncolytic virus is selected from the group consisting of an M1 virus, a Gaeta virus, a Sindbis virus, a Sendai virus, a Coxsackie virus, a herpes simplex virus, a parvovirus, an adenovirus, and an adeno-associated virus. , poliovirus, Newcastle disease virus, vesicular stomatitis virus, measles virus, reovirus, retrovirus, vaccinia virus, influenza virus and their engineered strains.
The use according to claim 28, wherein the Epac direct or indirect agonist is selected from the group consisting of a combination of one or more of a cAMP analog, an adenylate cyclase agonist, and a phosphodiesterase inhibitor.
The use according to claim 30, wherein said cAMP analog is selected from the group consisting of dibutyryl cyclic adenosine monophosphate (db-cAMP), 8-(4-thiochlorophenyl)-3', 5'-cyclic adenosine monophosphate (8‐CPT‐cAMP), 3′, 5′-cyclic adenosine monophosphate (Rp‐cAMPS), 2′-oxo-(2‐carbamoyl)‐3′, 5′-cyclic thio Adenosine phosphate (Rp‐2'‐AEC‐cAMPS/Rp‐2′‐EDA‐cAMPS), 8-‐(6-hexanediamino)‐3′, 5′-cyclic adenosine monophosphate (Rp‐8) ‐AHA‐cAMPS), agarose gel-fixed 8-(6-hexanediamino)-3',5'-cyclic adenosine monophosphate (Rp‐8‐AHA‐cAMPS‐Agarose), 8‐( 2-Ethylenediamine)-3',5'-cyclic adenosine monophosphate (Rp-8-AEA-cAMPS), 8-(2-ethanediamino)-2'-oxo-methyl- 3',5'-cyclic adenosine monophosphate (8-AET‐2'‐O‐Me‐cAMP), 8-‐(6-hexanediamino)‐2′‐oxo-methyl- 3′, 5' Cyclocyladenosine (8-AHA‐2'‐O‐Me‐cAMP), 8-benzylthio- 2′-oxo-methyl-3′, 5′-cyclic adenosine monophosphate (Sp‐ 8‐BnT‐2'‐O‐Me‐cAMPS/“S‐223”), 8-benzylthio-3', 5'-ring Adenosine Adenosine (Sp‐8‐BnT‐cAMPS/“S‐220”), 1‐N-oxy-3′, 5′-cyclic adenosine monophosphate (1‐NO‐cAMP), 3′, 5′ ‐ Cyclic adenosine monophosphate (cAMP), N‐6‐(2‐aminoethyl)‐3′, 5′-cyclic adenosine monophosphate (Sp‐6‐AE‐cAMPS), 8‐(2‐[DY‐ 547]-Aminoethylthio)-3',5'-cyclic adenosine monophosphate, 8-(6-hexanediamino)-2-chloro-3',5'-cyclic adenosine monophosphate (8-AHA- A combination of one or more of 2‐Cl‐cAMP) and 8-(6-hexanediamino)-3',5'-cyclic adenosine monophosphate (8-AHA-cAMP).
The use according to claim 30, wherein the adenylate cyclase agonist is selected from forskolin.
The use according to claim 30, wherein the phosphodiesterase inhibitor is selected from the group consisting of 3-isobutyl-1-methylxanthine (IBMX), Cilomilast, and Roflumilast (Roflumilast). ), Luteolin, Dyphylline, Rolipram, Sildenafil Citrate, Tadalafil, Vardenafil Hydrate hydrochloride (Vardenafil HCl Trihydrate), Pimobendan, GSK256066, PF-2552020, Apremilast (CC-10004), Cilostazol, Cipropirone (Cilostazol) Milrinone), one or more of Avanafil, Aminophylline, Dipyridamole, Doxofylline, and Deltarasin.
The use according to claim 28, wherein the tumor is a solid tumor or a hematoma.
The use according to claim 28, wherein the tumor is selected from the group consisting of liver cancer, colorectal cancer, bladder cancer, breast cancer, cervical cancer, prostate cancer, glioma, melanoma, pancreatic cancer, nasopharyngeal carcinoma, lung cancer, gastric cancer , ovarian cancer and leukemia.
The use according to claim 28 or 29 or 30, wherein the tumor is a tumor that is insensitive to the oncolytic virus.