CN105233285B

CN105233285B - The use in conjunction of Epac direct or indirect agonist and oncolytic virus

Info

Publication number: CN105233285B
Application number: CN201510563433.2A
Authority: CN
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: Guangzhou Weirongte Pharmaceutical Technology Co Ltd
Current assignee: GUANGZHOU WEIRONGTE PHARMACEUTICAL TECHNOLOGY CO., LTD.
Priority date: 2015-09-06
Filing date: 2015-09-06
Publication date: 2018-10-12
Anticipated expiration: 2035-09-06
Also published as: CN105233285A; WO2017036284A1

Abstract

The present invention provides a kind of pharmaceutical composition comprising the direct or indirect agonists of Epac and oncolytic virus, including the drug of the direct or indirect agonists of the Epac of independent packaging and the oncolytic virus of independent packaging is set with, the method of produced in vitro oncolytic virus, and Epac connects or tumour, the purposes in tumour especially insensitive to the oncolytic virus are being treated in the combination of agonist and oncolytic virus indirectly.The invention demonstrates that there is Epac agonists selectivity to increase oncolytic virus duplication effect, good security.Epac agonist energy selectively increasing tumor cell virus replications, and normal cell inner virus is replicated without influence, show that Epac agonists have tumor cells selectivity.

Description

The use in conjunction of Epac direct or indirect agonist and oncolytic virus

Technical field

The present invention relates to application of the use in conjunction of the direct or indirect agonists of Epac and oncolytic virus in treating tumour, In particular it relates to a kind of pharmaceutical composition comprising the direct or indirect agonists of Epac and oncolytic virus, including independent packaging The direct or indirect agonists of Epac and independent packaging oncolytic virus drug suit, the method for produced in vitro oncolytic virus, And Epac connects or tumour is being treated in the combination of agonist and oncolytic virus indirectly, it is especially insensitive to the oncolytic virus Purposes in tumour.

Background technology

Tumour is derived from the cumulative change of gene and epigenetics in normal cell, and this change drives normal cell to change For malignant tumour.This complicated pathological change process determine different tumours in generation, maintenance and transfer mechanism it is various Property.Currently, operation excision, chemotherapy and radiation are the common methods of clinical treatment tumour, however ocal resection easily recurs, Radiotherapy chemotherapy toxic side effect is big.The cancer of 15~20% mankind is related with viral infection, such as hepatitis type B virus (HBV), the third type liver Scorching virus (HCV) and liver cancer, human papilloma virus (HPV) and cervical carcinoma etc..

Oncolytic virus (oncolytic virus) is a kind of targeting infection and killing tumor cell, without destroying normally The replication-competent virus of cell.Oncolytic viral therapy (oncolytic virotherapy) is a kind of neoplasm targeted therapy of innovation Strategy, its using antiviral selectivity natural or through genetic engineering transformation infected tumor's cell, and it is multiple in tumour cell System has the function that targeting dissolving, killing tumor cell, but harmless to normal cell.

Present oncolytic virus treatment mainly faces following two problems.First, the Antitumor test of oncolytic virus is narrow：It is right The tumour cell of oncolytic virus sensitivity, along with more virus replication；The tumour cell insensitive to oncolytic virus, along with Less virus replication.Second, in vivo, over time, viral duplication can be restricted, and slowly clear by body It removes.Therefore, the duplication for being effectively increased oncolytic virus with how allowing tumor cells selectivity is problem in the urgent need to address.

Invention content

One aspect of the present invention provides a kind of pharmaceutical composition for treating tumour, and it is directly or indirectly exciting that it includes Epac Agent and oncolytic virus.Another aspect of the present invention provides the drug for treating tumour and is set with, and it includes the Epac of independent packaging is straight Connect or indirectly agonist and independent packaging oncolytic virus.

In one embodiment, the direct or indirect agonists of the Epac are selected from cAMP analogs, adenyl cyclase One or more combinations in agonist, phosphodiesterase inhibitors.The cAMP analogs are selected from Calcium Dibutyryladenosine Cyclophosph-ate acid gland Glycosides (db-cAMP), -3 ', 5 '-cyclic adenosine monophosphate (8-CPT-cAMP) of 8- (the thio chlorphenyls of 4-), 3 ', 5 '-cyclic phosphorothioic acid glands Glycosides (Rp-cAMPS), 2'- oxos-(2- ammonia second formoxyl) -3 ', 5 '-cyclic phosphorothioic acid adenosine (Rp-2'-AEC-cAMPS/Rp- 2'-EDA-cAMPS), -3 ', 5 '-cyclic phosphorothioic acid adenosines (Rp-8-AHA-cAMPS) of 8- (6- hexamethylene diamines base), Ago-Gel Fixed -3 ', 5 '-cyclic phosphorothioic acid adenosine (Rp-8-AHA-cAMPS-Agarose) of 8- (6- hexamethylene diamines base), 8- (2- ethylenediamines Base) -3 ', 5 '-cyclic phosphorothioic acid adenosines (Rp-8-AEA-cAMPS), 8- (2- ethylenediamines base) -2'- oxos-methyl -3 ', 5 '-rings Adenosine phosphate (8-AET-2'-O-Me-cAMP), 8- (6- hexamethylene diamines base) -2'- oxos-methyl -3 ', 5 '-cyclic adenosine monophosphate (8- AHA-2'-O-Me-cAMP), 8- benzylthios -2'- oxos-methyl -3 ', 5 '-cyclic phosphorothioic acid adenosine (Sp-8-BnT-2'-O- Me-cAMPS/ " S-223 "), 8- benzylthios -3 ', 5 '-cyclic phosphorothioic acid adenosines (Sp-8-BnT-cAMPS/ " S-220 "), 1-N- Oxygroup -3 ', 5 '-cyclic adenosine monophosphate (1-NO-cAMP), 3 ', 5 '-cyclic adenosine monophosphate (cAMP), N-6- (2- aminoethyls) -3 ', 5 ' - Cyclic phosphorothioic acid adenosine (Sp-6-AE-cAMPS), 8- (2- [DY-547]-ammonia ethylmercapto group) -3 ', 5 '-cyclic adenosine monophosphate, 8- (6- Hexamethylene diamine base) chloro- 3 ', the 5 '-cyclic adenosine monophosphate (8-AHA-2-Cl-cAMP) of -2- and 8- (6- hexamethylene diamines base) -3 ', 5 '-cycli phosphates One or more combinations in adenosine (8-AHA-cAMP).The adenyl cyclase agonist is selected from Forskolin.It is described Phosphodiesterase inhibitors are selected from 3-isobutyl-1-methylxanthine (IBMX), cilomilast (Cilomilast), roflumilast (Roflumilast), digicitrine (Luteolin), diprophylline (Dyphylline), rolipram (Rolipram), Sildenafil citrate (Sildenafil Citrate), Tadalafei (Tadalafil), three hydrochloride hydrate of Vardenafil (Vardenafil HCl Trihydrate), pimobendan (Pimobendan), GSK256066, PF-2545920, A Pusi Special (Apremilast (CC-10004)), Cilostazol (Cilostazol), Milrinone (Milrinone), avanaphil (Avanafil), aminophylline (Aminophylline), Dipyridamole (Dipyridamole), doxofylline (Doxofylline) With it is one or more in Deltarasin.

In one embodiment, the oncolytic virus is selected from M1 viruses, getah virus, sindbis alphavirus, celestial platform disease Poison, Coxsackie virus, herpes simplex virus, parvovirus, adenovirus, adeno-associated virus, poliovirus, newcastle disease Virus, vesicular stomatitis virus, measles virus, reovirus, retrovirus, vaccinia virus, influenza virus and they Engineered strain..In certain embodiments of the present invention, the oncolytic virus is M1 viruses or its engineered strain.

In one embodiment, the tumour is solid tumor or blood tumor.In one embodiment, the tumour choosing From liver cancer, colorectal cancer, carcinoma of urinary bladder, breast cancer, cervical carcinoma, prostate cancer, glioma, melanoma, cancer of pancreas, nasopharyngeal carcinoma, Lung cancer, gastric cancer, oophoroma and leukaemia.In some embodiments, the tumour is the tumour insensitive to oncolytic virus. In one embodiment, the tumour is the tumour insensitive to M1 oncolytic virus.

Another aspect of the present invention provides a kind of method producing oncolytic virus, including：Cultivate cell；In the cell of culture It is inoculated with oncolytic virus；The direct or indirect agonists of Epac are added into the cell of culture；Cell culture fluid is collected, is isolated and purified To the oncolytic virus.In one embodiment, the oncolytic virus be selected from M1 viruses, getah virus, sindbis alphavirus, 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, influenza virus and Their engineered strain.In one embodiment, the oncolytic virus is M1 viruses or its engineered strain.

Another aspect of the present invention provides a kind of method that promotion oncolytic virus replicates in tumour cell, including：To institute It states tumour cell and applies the oncolytic virus before, after or at the same time, the Epac of therapeutically effective amount is applied to the tumour cell Direct or indirect agonist.

Further aspect of the present invention provides a kind of method using object of the oncolytic virus treatment with tumour, including：To institute State the oncolytic virus that object applies therapeutically effective amount；In step (a) before, after or at the same time, apply to the object With the direct or indirect agonists of the Epac of therapeutically effective amount.

The invention demonstrates that the direct or indirect agonists of Epac can be used for assisting oncolytic virus treatment tumour.Cytologic experiment Prove that Epac agonists cause the oncolytic virus of kinds of tumor cells to replicate and increase；Zoopery proves Epac agonists specifically Increase the duplication of oncolytic virus within the tumor, to inhibit the growth of tumour.

The invention demonstrates that there is Epac agonists selectivity to increase oncolytic virus duplication effect, good security.Epac swashs Dynamic agent energy selectively increasing tumor cell virus replication, and normal cell inner virus is replicated without influence, show Epac agonists With tumor cells selectivity；In tumor bearing nude mice body, the Epac agonists through tail vein injection can selectively increase tumour Oncolytic virus amount is organized, and, the two virus quantity difference about 10 relatively low in normal structure inner virus amount²~10⁶Times, it furthers elucidate The tumor-selectives of Epac agonists.Epac agonists are given simultaneously and oncolytic virus (such as M1 viruses) has no effect on nude mice Weight and the state of mind show the two drug combination good security.

Description of the drawings

Fig. 1 cAMP Pathway Activations dramatically increase oncolytic virus protein expression；

A) cAMP Pathway Activations dramatically increase the table of oncolytic virus M1 structural proteins and non-structural protein in tumour cell It reaches；

B) cAMP Pathway Activations do not increase the expression of oncolytic virus M1 structural proteins and non-structural protein in normal cell.

Fig. 2 cAMP Pathway Activations dramatically increase oncolytic virus titre；

A) cAMP Pathway Activations increase the duplication of oncolytic virus M1 in tumour cell；

B) cAMP Pathway Activations do not increase the duplication of oncolytic virus M1 in normal cell.

Fig. 3 cAMP Pathway Activations significantly inhibit the expression of antiviral agent；

A-f) the expression of the infection induced antiviral agents of M1；

G-i) Epac agonists inhibit the expression of the antiviral agent of M1 inductions；

J-l) Epac agonists inhibit the expression of the antiviral agent of M1 inductions.

Fig. 4 cAMP Pathway Activations dramatically increase cytopathic effect caused by oncolytic virus M1；

A) cytopathic effect caused by cAMP Pathway Activations increase oncolytic virus M1 in tumour cell；

B) cAMP Pathway Activations do not increase cytopathic effect caused by oncolytic virus M1 in normal cell.

Fig. 5 cAMP Pathway Activations dramatically increase the anti-tumor effect of oncolytic virus；

A) cAMP Pathway Activations increase the oncolytic effect of oncolytic virus in tumour cell；

B) cAMP Pathway Activations do not increase the oncolytic effect of oncolytic virus in normal cell.

The other Epac agonists of Fig. 6 dramatically increase the anti-tumor effect of oncolytic virus；

A) Forskolin and 8-CPT-cAMP can also combine the growth that M1 significantly inhibits tumour cell；

B) Forskolin and 8-CPT-cAMP can also increase the titre of tumour cell.

The increased oncolytic effect of Fig. 7 cAMP Pathway Activations is rather than the PKA by Epac；

A) PKA interference cannot cancel the increased oncolytic effect of cAMP Pathway Activations；

B) Epac1 interference can cancel the increased oncolytic effect of cAMP Pathway Activations；

C) Epac1, rather than PKA, the increase of the virus protein of mediation.

Fig. 8 cAMP Pathway Activations increase the duplication of oncolytic virus in vivo；

CAMP Pathway Activations increase the duplication of oncolytic virus in tumor tissue specificity.QRT-PCR detection intravenous injections After cAMP Pathway Activations agent/M1 viruses, in the Tissue distribution of HCT116 mice with tumor.*, p<0.01.

Fig. 9 Epac agonists effectively inhibit mice with tumor tumour growth with oncolytic virus combination；

A) Epac agonists indicate M1 diseases with the influence to HCT116 mice with tumor gross tumor volumes after the intravenous injection of M1 viruses, M1 Malicious processing group, control group indicate OptiPRO^TMSFM culture medium solvent groups, cAMP indicate 8-CPT-cAMP processing groups.cAMP+M1 Indicate the viral combined processing groups of 8-CPT-cAMP and M1；

B) Epac agonists and the influence to Hep3B mice with tumor gross tumor volumes after the intravenous injection of M1 viruses,

C) Epac agonists and the influence to Capan-1 mice with tumor gross tumor volumes after the intravenous injection of M1 viruses,

Gross tumor volume is indicated with mean+SD, with the variance analysis statistical analysis of duplicate measurements；* indicates statistics It learns and examines p<0.01；I.v. tail vein injection is indicated.

Figure 10 Epac activation dramatically increases oncolytic virus titre in vehicles cells；

Db-cAMP dramatically increases duplications of the oncolytic virus M1 in different vehicles cells.

Specific implementation mode

As used herein, term " composition " refers to suitable for being applied to expected animal target to reach the preparation of therapeutic purposes, It contains at least one medicine activity component, such as compound.Optionally, the composition also contains at least one pharmaceutically Acceptable carrier or excipient.

Term is " pharmaceutically acceptable " to indicate that the substance does not have such characteristic, that is, considers the disease that will be treated Disease or illness and respective administration method, the Medical practitioners which will make rationality careful avoid taking the object to patient Matter.For example, for injectable objects, usually require that such substance is substantially sterile.

Herein, term " therapeutically effective amount " and the amount of " effective quantity " expression substance and substance are for preventing, subtracting One or more symptoms that are light or improving disease or illness, and/or it is effective to extend the survival for the object for receiving treatment.

I. oncolytic virus

Have a large amount of report displays, in vitro experiment, many viruses can replicate in kinds of tumor cells and cause to swell Oncocyte is dead, as sindbis alphavirus, sendai virus, Coxsackie virus, herpes simplex virus, parvovirus, adenovirus, Adeno-associated virus, poliovirus, newcastle disease virus, vesicular stomatitis virus, measles virus, reovirus, reverse transcription Virus, vaccinia virus and influenza virus.In addition, it was demonstrated that these viruses are effective to treating cancer animal model.

It is a kind of therapeutic strategy of novelty that oncolytic viral therapy, which compares for traditional remedies, and oncolytic viral therapy is to be based on The theory of selection or design antiviral selectivity replicated in tumour cell and cause death of neoplastic cells.In recent years, it grinds The person of studying carefully find successively some oncolytic virus using the immune deficiency of tumour-specific come selective killing tumor cell, wherein wrapping Include adenovirus, vaccinia virus, measles virus, vesicular stomatitis virus and herpes simplex virus etc..The clinic of these oncolytic virus Preceding research is also encouraging, can cause variety classes tumour regression.

In recent years, the extensive randomized clinical trial of oncolytic virus anti-cancer therapies is carrying out, such as in melanoma metastasis That carries out in patient can encode the III clinical trial phases of the attenuation HSV-1 viruses of the GM-CSF factors；It is recurred in head and neck neoplasm That carries out in patients examines the II clinical trial phases of reovirus and taxol and carboplatin combination effect；And it is directed to The II for comparing JX-594 therapeutic effects and supporting treatment therapeutic effect that the invalid liver cancer patient of Sorafenib treatments is carried out Phase randomized clinical trial etc..Whether these clinical tests compare deposits between oncolytic viral therapy and other traditional anti-cancer therapies It is acting synergistically, the immunoloregulation function and stimulation body for also exploring transgenosis oncolytic virus generate anti tumor immune response work( It can be for the effect of oncotherapy.

In the present invention, oncolytic virus can be natural or modified virus.Natural viral is, for example, M1 viruses, lid Tower virus, newcastle disease virus etc..Modified virus is also referred to as " engineered strain " in the present invention, is to utilize genetic engineering Etc. means various directional operations and transformation are carried out to viral gene, to be directionally altered and control behavior and the function of virus. Engineered purpose is carried out to natural oncolytic virus, including but not limited to, is weakened toxicity, is improved targeting, improve effect, increase Add safety etc..Engineered means include but not limited to gene mutation (such as rite-directed mutagenesis), knockout, exogenous sequences insert Enter, the missing etc. of redundancy segment.Existing most of oncolytic virus are modified virus, such as HSV-1 viruses, adenopathy Poison, vaccinia virus etc..

The cancer context that anticancer therapy is carried out using oncolytic virus includes solid tumor or blood tumor.In the present invention, may be used The cancer context that anticancer therapy is carried out using oncolytic virus is selected from liver cancer, colorectal cancer, carcinoma of urinary bladder, breast cancer, cervical carcinoma, forefront Gland cancer, glioma, melanoma, cancer of pancreas, nasopharyngeal carcinoma, lung cancer, gastric cancer, oophoroma and leukaemia.In some realities of the present invention It applies in mode, the tumour is the tumour insensitive to oncolytic virus.

II.Epac and its agonist

CAMP (cyclic adenosine monophosphate) is intracellular important one of second messenger, and ATP is catalyzed by adenyl cyclase (AC) It generates, can be degraded by phosphodiesterase.By changing intracellular cAMP concentration, signal in intracellular various kinds of cell is adjusted and turns Approach is led, to modulin activity, gene expression, realizes its physiological function.Many molecules such as prostaglandin are attached to cell The g protein coupled receptor on surface, activated adenyl cyclase, catalysis atriphos (ATP) generate cAMP, the intracellular ring of generation Adenosine phosphate can be degraded by phosphodiesterase, to make the signal path deactivate.The downstreams cAMP are mainly by cAMP according to lazyness Protein kinase A (PKA), the ion channel composition for guanylic acid exchanger (Epac) and the cAMP regulation and control that cAMP is relied on.

Epac is a kind of guanylic acid exchanger, has cAMP binding structural domains and guanylic acid switching fabric domain, can be by Intracellular second messenger cAMP activation plays many different biological actions, such as controls to activate the Rap albumen in downstream Adherency, invasion and the transfer of tumour cell.

It is an unexpected discovery of the invention that excitement Epac can specifically significantly increase oncolytic virus answering in tumour cell System, to make oncolytic virus cause death of neoplastic cells with higher efficiency.Inventor's discovery, the direct or indirect agonists of Epac Increase kinds of tumor cells the duplication of oncolytic virus.The direct or indirect agonists of Epac handle tumour cell with oncolytic virus 24,48 and 72 hours, duplication of the oncolytic virus in tumour cell is dramatically increased to time dependence.Therefore, when Epac excitements When agent is combined with oncolytic virus, the effect of oncotherapy can be significantly increased.

In the present invention, Epac agonists can be divided into Epac direct agonists and the indirect agonists of Epac by mechanism of action. In the present invention, Epac direct agonists are the groups for directly acting on Epac itself its compound or compound for expressing with excitement It closes；The indirect agonists of Epac refer to activating Epac ligands to the compound or compound combination of excitement Epac expression.

Therefore, cAMP and the like is Epac direct agonists, they are bonded directly to the cAMP integrated structures of Epac Domain, to the expression of excitement Epac.

CAMP analogs are any compounds for referring to effects of the simulation cAMP in the signal path, in the present invention It can be selected from one or more combinations in following cAMP analogs：Calcium Dibutyryladenosine Cyclophosph-ate (db-cAMP), 8- (4- sulphur For chlorphenyl) -3 ', 5 '-cyclic adenosine monophosphate (8-CPT-cAMP), 3 ', 5 '-cyclic phosphorothioic acid adenosines (Rp-cAMPS), 2'- oxygen Generation-(2- ammonia second formoxyl) -3 ', 5 '-cyclic phosphorothioic acid adenosines (Rp-2'-AEC-cAMPS/Rp-2'-EDA-cAMPS), 8- (6- Hexamethylene diamine base) -3 ', 5 '-cyclic phosphorothioic acid adenosines (Rp-8-AHA-cAMPS), Ago-Gel fixed 8- (6- hexamethylene diamines Base) -3 ', 5 '-cyclic phosphorothioic acid adenosines (Rp-8-AHA-cAMPS-Agarose), 8- (2- ethylenediamines base) -3 ', 5 '-epithio generations Adenosine phosphate (Rp-8-AEA-cAMPS), 8- (2- ethylenediamines base) -2'- oxos-methyl -3 ', 5 '-cyclic adenosine monophosphate (8-AET- 2'-O-Me-cAMP), 8- (6- hexamethylene diamines base) -2'- oxos-methyl -3 ', 5 '-cyclic adenosine monophosphate (8-AHA-2'-O-Me- CAMP), 8- benzylthios -2'- oxos-methyl -3 ', 5 '-cyclic phosphorothioic acid adenosine (Sp-8-BnT-2'-O-Me-cAMPS/ " S- 223 "), 8- benzylthios -3 ', 5 '-cyclic phosphorothioic acid adenosines (Sp-8-BnT-cAMPS/ " S-220 "), 1-N- oxygroups -3 ', 5 '-rings Adenosine phosphate (1-NO-cAMP), 3 ', 5 '-cyclic adenosine monophosphate (cAMP), -3 ', 5 '-cyclic phosphorothioic acid glands of N-6- (2- aminoethyls) Glycosides (Sp-6-AE-cAMPS), 8- (2- [DY-547]-ammonia ethylmercapto group) -3 ', 5 '-cyclic adenosine monophosphate, 8- (6- hexamethylene diamines base) -2- Chloro- 3 ', 5 '-cyclic adenosine monophosphate (8-AHA-2-Cl-cAMP) and 8- (6- hexamethylene diamines base) -3 ', 5 '-cyclic adenosine monophosphate (8-AHA- cAMP)。

The indirect agonist of Epac is, for example, that can increase the reagent of intracellular cAMP amount, such as adenyl cyclase The inhibitor etc. of agonist or phosphodiesterase.

Adenyl cyclase agonist can exciting adenyl cyclase, promote to generate cAMP by ATP, in the present invention, It can be selected from Forskolin (Forskolin).

Phosphodiesterase inhibitors can inhibit degradation of the phosphodiesterase to cAMP, it can be selected from the present invention：3- is different Butyl -1- methyl xanthines (IBMX), cilomilast (Cilomilast), roflumilast (Roflumilast), digicitrine (Luteolin), diprophylline (Dyphylline), rolipram (Rolipram), sildenafil citrate (Sildenafil Citrate), Tadalafei (Tadalafil), three hydrochloride hydrate of Vardenafil (Vardenafil HCl Trihydrate), pimobendan (Pimobendan), GSK256066, PF-2545920, Apremilast (Apremilast (CC- 10004)), Cilostazol (Cilostazol), Milrinone (Milrinone), avanaphil (Avanafil), aminophylline (Aminophylline), in Dipyridamole (Dipyridamole), doxofylline (Doxofylline) and Deltarasin It is one or more.

III. pharmaceutical composition and application

In the present invention, " pharmaceutical composition " refers to including the composition of compound and oncolytic virus, wherein compound and Oncolytic virus is present in composition in hybrid form.The composition will generally be used for the treatment of human subjects.However, it Can also be used to treat the similar or identical illness in other animal targets.Herein, term " object ", " animal target " and similar terms refer to people and non-human vertebrate, such as mammal, such as non-human primates, sport animals And commercial animal, such as horse, ox, pig, sheep, rodent and pet (such as dog and cat).

Suitable dosage form, is partly dependent on purposes or the approach of administration, for example, orally, percutaneously, transmucosal, sucking or logical Cross injection (parenteral).Such dosage form should enable the compound/oncolytic virus reach target cell.Other factors are in this field In be well known, including consideration, such as toxicity and delay compound or composition play the dosage form of its effect.Skill Art and formula generally can in The Science and Practice of Pharmacy, 21^st edition, It is found in Lippincott, Williams and Wilkins, Philadelphia, PA, 2005 (being hereby incorporated by reference).

Carrier or excipient can be used to produce composition.The carrier or excipient can be selected as promoting chemical combination The administration of object.The example of carrier includes calcium carbonate, calcium phosphate, various sugared (such as lactose, glucose or sucrose) or starch Type, cellulose derivative, gelatin, vegetable oil, polyethylene glycol and physiological compatibility solvent.The example packet of physical compatibility solvent Include water for injection (WFI) sterile solution, salting liquid and glucose.

Can apply the component of composition or composition by different paths, including in intravenous, peritonaeum, subcutaneous, flesh Interior, oral, transmucosal, rectum, percutaneous or sucking.In some embodiments, it is preferred injection or freeze drying powder injection.To oral For, for example, compound can be configured to conventional oral dosage formulations, such as capsule, tablet and liquid preparation, such as syrup, Elixir and inspissated drops.

For inhalant, the composition of the present invention or its component can be formulated as dry powder or suitable solution, suspension Or aerosol.Powder and solution can be prepared with suitable additive known in the art.For example, powder may include suitable Powdered substrate (powder base) such as lactose or starch, solution may include propylene glycol, sterile water, ethyl alcohol, sodium chloride and other Additive such as acid, alkali and buffer salt.This solution can be given by sucking or hang through spraying, pump, sprayer or atomizer etc. Supernatant liquid..

The pharmaceutical preparation of oral use can be obtained, such as by combining composition or its component with solid excipient, Optionally grinding is formed by mixture, and the mixture of processing particle (is such as needed) after suitable adjuvant is added, to Obtain tablet or dragee.Suitable excipient is in particular, filler is for example sugared, including lactose, sucrose, mannitol or sorb Alcohol；Cellulose preparation, such as cornstarch, wheaten starch, rice starch, potato starch, gelatin, gum tragacanth, methyl are fine Tie up element, hydroxypropyl methyl cellulose, sodium carboxymethylcellulose (CMC) and/or polyvinylpyrrolidone (PVP：Povidone (povidone)).If desired, disintegrant can be added, for example, crosslinked polyvinylpyrrolidone, agar or alginic acid or they Salt, such as mosanom.

Alternatively, injection (parenteral administration), such as intramuscular, intravenous, in peritonaeum and/or skin can be used Under.For injection, composition of the invention or its component are configured to sterile aqueous solutions, preferably in PHYSIOLOGICALLY COMPATIBLE In buffer solution or solution, such as saline solution, Hank solution or Ringer solution.In addition, composition or its component can by with It is made as solid form, and a moment is redissolved or is suspended before the use.Freeze-dried powder form can also be produced.

Administration can also pass through transmucosal, part or transcutaneous modalities.For transmucosal, locally or percutaneously, be formulated It is middle to use the penetrating agent for being suitble to barrier to be penetrated.Such penetrating agent is generally known in the art, including, for example, For mucosal, bile salt and fusidic acid derivatives.In addition, detergent can be used for promoting penetrating.Mucosal, For example, nose spray or suppository (per rectum or vagina) can be passed through.

The topical formulations of the present invention can be preferably by selecting suitable carrier known in the art to be configured to oil, frost Agent, lotion, paste etc..If desired, the system of emulsifier, stabilizer, wetting agent and antioxidant and imparting color or fragrance Agent can also be included.The creme of local application is preferably prepared from the mixture of mineral oil, self emulsifying beeswax and water.This Outside, it includes transdermal patch or dressing, such as one kind or more known in the art equipped with active constituent and optionally that transdermal means, which are applied, The bandage of kind carrier or diluent.In order to be applied in the form of transdermal delivery system, the administration dosage during dosage regimen will be certain It is lasting, rather than intermittent.

The effective quantity of various components to be administered, for example described compound of factor of consideration can be determined by standardization program IC₅₀, the biological half-life of the compound, age, size and the weight of object and illness related with object.These factors Importance with other factors is well known for those of ordinary skill in the art.In general, dosage will be treated The about 0.01mg/kg of object is between 50mg/kg, preferably in 0.lmg/kg between 20mg/kg.Multiple agent can be used Amount.As optional administering mode, Epac agonists of the present invention can be administered by vein or intratumor injection mode.Intratumor injection 5mg/kg~500mg/kg is given once daily in mode；5mg/kg~5g/kg is given once daily in a manner of intravenous injection.

The composition of the present invention or its component can also be used in combination with the other therapeutic agents for the treatment of same disease.This knot It closes use and is included in different time using these compounds and oncolytic virus and one or more other therapeutic agents, or make simultaneously With this compound and oncolytic virus and one or more other therapeutic agents.It in some embodiments, can be to the one of the present invention Kind or multiple compounds/oncolytic virus or the dosage of the other therapeutic agents of combined use are modified, for example, passing through this field skill Method known to art personnel is reduced relative to the compound of exclusive use or the dosage of therapeutic agent.

It is to be understood that be used in combination or combination include be used together with other therapies, drug, medical procedures etc., wherein Other therapies or program can be in the times different from composition or its component of the invention (for example, (such as several small in a short time When, such as 1,2,3,4-24 hour) in a long time (such as 1-2 days, 2-4 days, 4-7 days, 1-4 weeks) or with group of the invention It closes object or its component identical time is administered.Combined use further includes and therapy or medical procedures primary or infrequently apply (as performed the operation) is used together, and short before or after other therapies or program with the composition or its component of the present invention Application in phase or longer period.In some embodiments, the present invention is used to deliver the composition or its component of the present invention With one or more other drugs therapeutic agents, they are delivered by identical or different administration route.

Any administration route combination application include by identical administration route by the present invention composition or its component and One or more other drugs therapeutic agents are delivered together with any dosage form, including two kinds of compounds are chemically connected and they The preparation of respective therapeutic activity is kept in application.In one aspect, the other drugs therapy can with the present invention composition or Its component is co-administered.By the combined use of co-administration including the total preparation (co-formulation) of application or chemically The preparation of the compound of connection, or in a short time (for example, in hour, in 2 hours, in 3 hours, until in 24 hours) apply With the compound of two or more independent formulations forms, they are administered with identical or different approach.

The co-administration of independent formulations include via the co-administration of the delivering of a device, such as identical suction apparatus, Identical syringe etc., or applied in a short time by different device relative to each other.Pass through the change for the present invention that identical administration route delivers The total preparation for closing object and one or more additional medicinal treatments includes preparing material together to which they can be filled by one It sets and is administered, including different compound combinations are in a kind of preparation or compound is modified so that they connect in chemistry It is connected together but still keeps respective biological activity.This compound chemically connected may include two active constituents point The connector opened, the connector maintain substantially in vivo, or in vivo may degradation.

IV. drug is set with

In the present invention, " drug suit " refer to comprising independent packaging oncolytic virus drug and independent packaging Epac it is straight It connects or the Key works Drug packing of agonist drug indirectly, wherein oncolytic virus and agonist respectively exists with suitable dosage form, to just In by the two separate administration at certain intervals.In some embodiments, described " drug suit " also may include operation instructions, Application instrument.

" independent packaging " refers to separately made dosage form in the present invention.For example, in drug suit of the invention In one embodiment, oncolytic virus exists in the form of single dose injection agent, and Epac agonists exist in form of tablets.At this In the another embodiment of invention, oncolytic virus exists in the form of freeze-dried powder, and Epac agonists are with the shape of injection Formula exists.Those skilled in the art are it is contemplated that according to the respective property of oncolytic virus and Epac agonists, the two can be distinguished It is made into other suitable dosage forms, to be packed jointly into identical Key works Drug packing, to form drug of the present invention Suit.

The oncolytic virus drug of independent packaging and the direct or indirect agonist drugs of the Epac of independent packaging are respectively with suitable Dosage be present in the present invention drug suit in.The dosage can be the combination of single dosage or multiple dosage, so that finally It is applied to object with the dosage of therapeutically effective amount.Specific dosage can be come by those skilled in the art according to routine test and experience true It is fixed.

V. method

In one aspect, the present invention provides a kind of method that promotion oncolytic virus replicates in tumour cell, including：To The tumour cell applies the oncolytic virus before, after or at the same time, and therapeutically effective amount is applied to the tumour cell The direct or indirect agonists of Epac.

Another aspect of the invention provides a kind of method using object of the oncolytic virus treatment with tumour, including：(a) The oncolytic virus of therapeutically effective amount is applied to the object；(b) it is carried out before, after or at the same time, to institute in step (a) State the direct or indirect agonists of Epac that object applies therapeutically effective amount.

In one embodiment, the oncolytic virus of therapeutically effective amount is applied to object first, then to the object Using the direct or indirect agonists of the Epac of therapeutically effective amount.In another embodiment, have first to object using treatment The direct or indirect agonists of Epac of effect amount, then to the object using the oncolytic virus of therapeutically effective amount.At another In embodiment, the direct or indirect agonists of Epac and therapeutically effective amount of therapeutically effective amount are applied to object at the same time The oncolytic virus.

The application of oncolytic virus and agonist can be spaced the short time (such as several hours, such as 1,2,3,4-24 hours) or Every longer time (such as 1-2 days, 2-4 days, 4-7 days, 1-4 weeks).

Another aspect of the invention provides a kind of method producing oncolytic virus, including：Cultivate cell；In the thin of culture Oncolytic virus is inoculated in born of the same parents；The direct or indirect agonists of Epac are added into the cell of culture；And cell culture fluid is collected, point The oncolytic virus is obtained from purifying.In certain embodiments of the present invention, the cell is selected from Vero cells, Chinese hamster ovary celI With HEK293 cells.

In the method, the oncolytic virus is selected from M1 viruses, getah virus, sindbis alphavirus, sendai virus, Ke Sa Qi viruses, herpes simplex virus, parvovirus, adenovirus, adeno-associated virus, poliovirus, newcastle disease virus, Vesicular stomatitis virus, measles virus, reovirus, retrovirus, vaccinia virus, influenza virus and their engineering change Make strain.

In the method, the tumour is solid tumor or blood tumor.The wherein described tumour be selected from liver cancer, colorectal cancer, Carcinoma of urinary bladder, breast cancer, cervical carcinoma, prostate cancer, glioma, melanoma, cancer of pancreas, nasopharyngeal carcinoma, lung cancer, gastric cancer, oophoroma Or leukaemia.Preferably, the tumour is the tumour insensitive to oncolytic virus.

In the method, the direct or indirect agonists of the Epac are selected from cAMP analogs, adenyl cyclase excitement One or more combinations in agent, phosphodiesterase inhibitors.The cAMP analogs are selected from Calcium Dibutyryladenosine Cyclophosph-ate (db-cAMP), -3 ', 5 '-cyclic adenosine monophosphate (8-CPT-cAMP) of 8- (the thio chlorphenyls of 4-), 3 ', 5 '-cyclic phosphorothioic acid adenosines (Rp-cAMPS), 2'- oxos-(2- ammonia second formoxyl) -3 ', 5 '-cyclic phosphorothioic acid adenosine (Rp-2'-AEC-cAMPS/Rp-2'- EDA-cAMPS), -3 ', 5 '-cyclic phosphorothioic acid adenosines (Rp-8-AHA-cAMPS) of 8- (6- hexamethylene diamines base), Ago-Gel are fixed - 3 ', 5 '-cyclic phosphorothioic acid adenosines (Rp-8-AHA-cAMPS-Agarose) of 8- (6- hexamethylene diamines base), 8- (2- ethylenediamines base)- 3 ', 5 '-cyclic phosphorothioic acid adenosines (Rp-8-AEA-cAMPS), 8- (2- ethylenediamines base) -2'- oxos-methyl -3 ', 5 '-ring phosphorus Adenosine monophosphate (8-AET-2'-O-Me-cAMP), 8- (6- hexamethylene diamines base) -2'- oxos-methyl -3 ', 5 '-cyclic adenosine monophosphate (8- AHA-2'-O-Me-cAMP), 8- benzylthios -2'- oxos-methyl -3 ', 5 '-cyclic phosphorothioic acid adenosine (Sp-8-BnT-2'-O- Me-cAMPS/ " S-223 "), 8- benzylthios -3 ', 5 '-cyclic phosphorothioic acid adenosines (Sp-8-BnT-cAMPS/ " S-220 "), 1-N- Oxygroup -3 ', 5 '-cyclic adenosine monophosphate (1-NO-cAMP), 3 ', 5 '-cyclic adenosine monophosphate (cAMP), N-6- (2- aminoethyls) -3 ', 5 ' - Cyclic phosphorothioic acid adenosine (Sp-6-AE-cAMPS), 8- (2- [DY-547]-ammonia ethylmercapto group) -3 ', 5 '-cyclic adenosine monophosphate, 8- (6- Hexamethylene diamine base) chloro- 3 ', the 5 '-cyclic adenosine monophosphate (8-AHA-2-Cl-cAMP) of -2- and 8- (6- hexamethylene diamines base) -3 ', 5 '-cycli phosphates Adenosine (8-AHA-cAMP).The adenyl cyclase agonist is selected from Forskolin (Forskolin).The phosphodiesterase Inhibitor is selected from 3-isobutyl-1-methylxanthine (IBMX), cilomilast (Cilomilast), roflumilast (Roflumilast), digicitrine (Luteolin), diprophylline (Dyphylline), rolipram (Rolipram), Sildenafil citrate (Sildenafil Citrate), Tadalafei (Tadalafil), three hydrochloride hydrate of Vardenafil (Vardenafil HCl Trihydrate), pimobendan (Pimobendan), GSK256066, PF-2545920, A Pusi Special (Apremilast (CC-10004)), Cilostazol (Cilostazol), Milrinone (Milrinone), avanaphil (Avanafil), aminophylline (Aminophylline), Dipyridamole (Dipyridamole), doxofylline (Doxofylline) With it is one or more in Deltarasin.Preferably, the application is intravenous injection.

1 Epac agonists of embodiment dramatically increase the duplication of oncolytic virus.

1) Epac agonists dramatically increase the expression of oncolytic virus albumen.

Material：

People's Colon and rectum cell cancer HCT116, human pancreas' cell cancer Capan-1, people immortalize normal liver cell strain L-02, M1 Virus, DMEM in high glucose culture medium, db-cAMP (Sigma, USA).

Western bolt：Total protein of cell extract (Mammalian Protein Extraction Reagent, Thermo), E1 antibody (self-control), NS3 antibody (self-control), GAPDH antibody (Bioworld USA)；

Method：

A) culture of cell：People Colon and rectum cell carcinoma strain HCT116, human pancreas cell carcinoma strain Capan-1, people Normal liver cell strain L-02 is immortalized, it is complete to be grown in the DMEM containing 10%FBS, 100U/ml penicillin and 0.1mg/ml streptomysins In full culture medium or primitive cell culture base；All cell strains are placed in 5%CO₂, 37 DEG C of constant-temperature enclosed formula incubators are (relatively wet Subculture in 95%) is spent, inverted microscope observes growing state.Passage in about 2~3 days is primary, takes in exponential phase Cell for formally test.

B) virus protein detects：Select exponential phase cell, DMEM complete culture solutions (containing 10% fetal calf serum, 1% pair It is anti-) cell suspension is made, cell is with 3.0 × 10⁵The density of/ware is seeded in 10mm culture dishes.With db-cAMP (1mM)/M1 diseases Malicious (MOI=10) handled cell after 24 hours, the expression of western blot detection virus proteins.

As a result：

As shown in Figure 1a, db-cAMP processing dramatically increases expression to M1 virus proteins in tumour cell.Wherein control group with Db-cAMP groups individually are given, do not detect virus protein；Individually still less viral egg can be observed in infection M1 virus groups White expression；Compared with individually infection M1 virus groups, joint db-cAMP and M1 groups dramatically increase M1 virus structural proteins E1 and structure Albumen NS3 expressions.In addition, db-cAMP and M1 Combined Treatments cannot increase M1 viral protein expressions in normal cell.Such as Shown in Fig. 1 b, normal liver cell system L-02 cells are immortalized using the M1 viruses infection people of identical titre, whether individually infection M1 groups or joint db-cAMP and M1 groups, all expression without finding virus protein.The above result shows that activation cAMP accesses M1 virus replications can selectively be promoted in tumour cell, to increase the expression of oncolytic virus M1 albumen.

2) increase to Epac Agonist selectivities the titre of oncolytic virus M1

Material：

People's Colon and rectum cell cancer HCT116, human pancreas' cell cancer Capan-1, people immortalize normal liver cell strain L-02, M1 Virus, DMEM in high glucose culture medium, db-cAMP (Sigma, USA), BHK-21 cell strains.M1 virus titers measure (TCID50 methods):

(1) BHK-21 cells are cultivated, logarithmic growth phase cell is inoculated into 96 orifice plates, and cell suspension 100 is added per hole μ l, make cell concentration reach 2~3 × 10⁵A/ml.

(2) M1 virus liquids are made into continuous 10 times of dilution, from 10^-1-10^-10。

(3) by the virus inoculation diluted to 96 hole microtest plates, each dilution is inoculated with a tandem totally 8 hole, often Hole is inoculated with 20 μ l.

(4) normal cell controls are set, normal cell controls make two tandems.(+100 μ l cell suspensions of 20 μ l growth-promoting medias)

(5) it observes and records day by day as a result, generally requiring observation 5-7 days.

(6) calculating of result, by Reed-Muench Liang Shi method or Karber methods.

TCID50 computational methods

(1) Reed-Muench Liang Shi methods

CPE：Cytopathic effect

Distance proportion=(percentage -50% for being higher than 50% lesion rate)/(percentage-higher than 50% lesion rate is less than The percentage of 50% lesion rate)=(91.6-50)/(91.6-40)=0.8

Difference between lgTCID50=distance proportions × dilution logarithm+higher than 50% lesion rate dilution logarithm= 0.8 × (- 1)+(- 3)=- 3.8

TCID50=10^-3.8/0.1ml

Meaning：By the viral dilution 10^3.8100 μ l of inoculation can make 50% cell that lesion occur again.

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

(2) Karber methods

LgTCID50=L-d (s-0.5)

L：The logarithm of highest dilution (this example is -1)

D：Difference between dilution logarithm (this example is -1)

S：Positive hole ratio summation

LgTCID50=-1-1 × (3.375-0.5)=- 3.875

TCID50=10^-3.875/0.1ml

Meaning：By the viral dilution 10^3.875Times, 100 μ l of inoculation can make 50% cell that lesion occur.

Virus titer is：10^3.875TCID50/0.1ml=7.5 × 10⁴TCID50/ml

Method：

A) inoculating cell, administration processing：Select exponential phase cell, DMEM complete culture solutions (containing 10% fetal calf serum, 1% is dual anti-) cell suspension is made, with every hole 4 × 10³The density in/hole is seeded in 96 well culture plates.See that cell is complete after 12 hours Complete adherent, experiment divides M1 individually to organize and db-cAMP/M1 combination groups, experimental group：M1 virus (MOI=0.1) infection cells；Combination Group：Db-cAMP (500 μM) and M1 viral (MOI=0.1) handles cell.Collect cell conditioned medium within 0,24,48,72 hour.

B) prior kind of good BHK-21 cell is used, according to viral in the method gradient dilution detection supernatant of TCID50 Titre.

As a result:

As shown in Figure 2 a, individually infection can be in human pancreatic cancer cell Capan-1 and Human colorectal cancer cells for M1 viruses It is the increase of time dependence in HCT116.And the increasing of time dependence is unable in people immortalizes normal liver cell system L-02 Add virus quantity.At 24,48,72 hours, db-cAMP processing can significantly increase oncolytic virus M1 in human pancreatic cancer cell Duplication in Capan-1 and Human colorectal cancer cells system HCT116, and cannot significantly increase oncolytic virus M1 and be immortalized in people Duplication (Fig. 2 b) in normal liver cell system L-02.The increase oncolytic virus M1 that db-cAMP selectivity is further illustrated exists Duplication in tumour cell.

3) Epac1 agonists significantly inhibit the expression of the antiviral agent of M1 inductions.

Material：

People's Colon and rectum cell cancer HCT116, M1 virus, DMEM in high glucose culture medium, db-cAMP (Sigma, USA).RNA is extracted Reagent Trizol, real-time fluorescence quantitative PCR instrument.

Method：

A) culture of cell：People Colon and rectum cell carcinoma strain HCT116, is grown in containing 10%FBS, 100U/ml penicillin And in the DMEM complete mediums of 0.1mg/ml streptomysins；It is placed in 5%CO₂, 37 DEG C of constant-temperature enclosed formula incubator (relative humidity 95%) subculture in, inverted microscope observe growing state.Passage in about 2~3 days is primary, takes in exponential phase Cell is for formally testing.

B) antiviral agent is expressed：Exponential phase cell is selected, DMEM complete culture solutions are (containing 10% fetal calf serum, 1% It is dual anti-) cell suspension is made, cell is with 3.0 × 10⁵The density of/ware is seeded in 10mm culture dishes.With db-cAMP (1mM)/M1 After the time point specified in viral (MOI=10) processing cytological map, real-time quantitative RT-PCR detects the mRNA tables of antiviral agent Up to situation.

As a result：

As shown in Fig. 3 a-f, after M1 infects HCT116 colorectal cancer cells systems, illustrate time dependence induction these The expression of antiviral agent, including interferon-' alpha ' (IFNA), interferon beta (IFNB), interferon regulatory factor -3 (IRF-3) are done Disturb plain regulatory factor -7 (IRF-7) and interferon-induced gene (ISGs) MDA5 and IFIT1.As shown in Fig. 3 g-i, when us After infection M1 viruses 12 hours, Epac agonists db-cAMP and Forskolin can significantly inhibit these of M1 inductions The expression of antiviral agent.

2 Epac agonists of embodiment cause death of neoplastic cells with M1 antiviral selectivities

1) Epac agonists cause tumour cell lesion with M1 antiviral selectivities

Material：

People Colon and rectum cell cancer HCT116, people's immortalization normal liver cell strain L-02, M1 virus, DMEM in high glucose culture medium, Inverted phase contrast microscope.

Method：

A) culture of cell：People Colon and rectum cell cancer HCT116, people immortalize normal liver cell strain L-02, are grown in and contain In the DMEM complete mediums of 10%FBS, 100U/ml penicillin and 0.1mg/ml streptomysins；All cell strains are placed in 5% CO₂, 37 DEG C of interior subcultures of constant-temperature enclosed formula incubator (relative humidity 95%), inverted microscope observation growing state.About 2~ Passage in 3 days is primary, takes the cell in exponential phase for formally testing.

B) it is observed under microcytoscope：Select exponential phase cell, DMEM complete culture solutions (containing 10% fetal calf serum, 1% is dual anti-) cell suspension is made, cell is with 2.5 × 10⁴The density in/hole is seeded in 24 well culture plates.With M1 viruses (MOI= 1) after infecting 48 hours, the variation of cytomorphology is observed under inverted phase contrast microscope.

As a result：

As shown in fig. 4 a, cellular morphology is observed under phase contrast microscope, HCT116 cells and Capan-1 cells are single layer patches Wall is grown, and cell tight arranges, and phenotype is consistent.With the independent independent processing group of processing group and db-cAMP of cellular control unit, M1 Compare, after viral (MOI=1) the Combined Treatment 72h of db-cAMP and M1, cell number significantly reduces, and the form hair of cell It has given birth to and has substantially change, cell space shrinks glomeration, and index of refraction is remarkably reinforced, in dead lesion sample.As shown in Figure 4 b, db-cAMP joins Close M1 virus treateds does not have Morphology Effects to normal cell.Using the db-cAMP of same concentration and the M1 viruses of same titre It infects people and immortalizes normal liver cell system L-02 cells, do not find cell number and form significant change.Db- described above CAMP joint M1 virus treateds selectively dramatically increase death of neoplastic cells form lesion, and are not influenced on normal cell.

2) Epac agonists reduce cells survival rate with the viral combined processing of M1

Material：

People's Colon and rectum cell cancer HCT116, human pancreas' cell cancer Capan-1, people immortalizes normal liver cell strain L-02, M1 Virus, DMEM in high glucose culture medium, inverted phase contrast microscope.

Method：

A) inoculating cell, administration processing：Select exponential phase cell, DMEM complete culture solutions (containing 10% fetal calf serum, 1% is dual anti-) cell suspension is made, with every hole 4 × 10³The density in/hole is seeded in 96 well culture plates.See that cell is complete after 12 hours Complete adherent, experiment divides control group, independent db-cAMP groups, M1 infected groups and db-cAMP/M1 combination groups.Dosage used is：M1 diseases Malicious (MOI=10) infection cell；Db-cAMP sets different dose gradients.

B) MTT is reacted with intracellular succinate dehydrogenase：When culture is to 72h, 20 μ l (5mg/ml) of MTT are added per hole, Continue to be incubated 4 hours, the graininess bluish violet formazan crystallization that microscopy can be observed, be formed in living cells at this time.

C) Rong Xie formazans particle：Supernatant carefully is sucked, adds the 100 lysigenous crystallizations in the holes μ l/ of DMSO, in micro-oscillating 5min is shaken on device, then detects the optical density (OD values) in each hole with wavelength 570nm in enzyme detector.Every group of experiment repeats 3 times.Cell survival rate=drug-treated group OD values/control group OD value × 100%.

As a result:

As shown in Figure 5 a, individually processing there is M1 viruses smaller survival rate to press down tumour cell HCT116 and Capan-1 It makes and uses, cells survival inhibition is significantly caused after db-cAMP joint M1 processing.Such as Fig. 5 b, people immortalizes normal cell L- In 02, same db-cAMP joints M1 processing processing does not cause apparent cells survival rate to inhibit.It these results suggest that, db-cAMP Joint M1 virus treateds selectively significantly reduce tumor cell survival, and are not influenced on normal cell.

As shown in Figure 6 a, in order to further verify influence of the Epac activation to oncolytic virus M1 oncolytic effects, we continue Two kinds of Epac agonists of 8-CPT-cAMP and Forskolin are used, it is found that they combine M1 virus treateds and can also significantly draw The existence for playing cell inhibits.As shown in Figure 6 b, the two Epac agonists can also significantly increase the duplication of oncolytic virus M1.

The oncolytic effect of 3 Epac agonists of embodiment enhancing M1 is rather than the PKA by Epac1

1) interference PKA cannot cancel the oncolytic effect of cAMP activation enhancings

Material：

People's Colon and rectum cell cancer HCT116, M1 virus, DMEM in high glucose culture medium, MTT, siRNA, RNAiMAX, inverted phase contrast Microscope.

Method：

A) culture of cell：People Colon and rectum cell cancer HCT116, be grown in containing 10%FBS, 100U/ml penicillin and In the DMEM complete mediums of 0.1mg/ml streptomysins；Cell is placed in 5%CO₂, 37 DEG C of constant-temperature enclosed formula incubator (relative humidity 95%) subculture in, inverted microscope observe growing state.Passage in about 2~3 days is primary, takes in exponential phase Cell is for formally testing.

B) RNA is interfered：Select exponential phase cell, DMEM complete culture solutions (containing 10% fetal calf serum, 1% dual anti-) system At cell suspension, cell is with 2.5 × 10⁴The density in/hole is seeded in 24 well culture plates.It changes into without dual anti-, contains 10% tire ox blood Clear DMEM culture mediums are siRNA using RNAiMAX transfections 50nm.Out of order RNA is as a control group.Interference changes into after 24 hours DMEM complete culture solutions simultaneously infect M1 viruses, and MTT detects cells survival rate after 72 hours.

As a result：

As shown in Figure 7a, after HCT116 cells are transferred to out of order siRNA, with control group and individually processing db-cAMP or M1 groups compare, and db-cAMP/M1 is combined the survival rate that can significantly inhibit cell, that is to say, that db-cAMP is significantly enhanced The oncolytic effect of M1 viruses.After interfering PKA respectively using three different siRNA fragments, in HCT116 cells and control group Individually for processing group compared with db-cAMP individually processing group, db-cAMP/M1 Combined Treatments still can inhibit tumour thin by cell, M1 The growth of born of the same parents.As shown in Figure 7 c, PKA interference fragments have preferable jamming effectiveness, but it is increased to interfere PKA that cannot but block Virus protein.

2) interference Epac1 cancels the oncolytic effect of cAMP activation enhancings

Material：

People's Colon and rectum cell cancer HCT116, M1 virus, DMEM in high glucose culture medium, MTT, siRNA, RNAiMAX are inverted phase Poor microscope.

Method：

As a result：

As shown in Figure 7b, after HCT116 cells are transferred to out of order siRNA, with control group and individually processing db-cAMP or M1 groups compare, and db-cAMP/M1 is combined the survival rate that can significantly inhibit cell, that is to say, that db-cAMP is significantly enhanced The oncolytic effect of M1 viruses.After interfering Epac1 respectively using three different siRNA fragments, in HCT116 cells, db- CAMP/M1 Combined Treatments group cannot significantly inhibit the survival rate of cell.As shown in Figure 7 c, Epac1 interference fragments have preferable Jamming effectiveness, and interfere Epac1 that can block increased virus protein.

The viral combined processing of 4 Epac agonists of embodiment/M1 effectively inhibits tumour growth

1) Epac Agonist selectivities increase M1 virus replications in tumor tissues

Material：

4 week old female BAl BIcs/c nude mices, Human colorectal cancer cells system HCT116, RNA extracts reagent Trizol, tissue homogenate Machine, real-time fluorescence quantitative PCR instrument

Method：

To the nude mice dorsal subcutaneous of 4 week old injection 5 × 10⁶HCT116 cells.After 6 days, every mouse tail vein injects M1 Virus (3 × 10⁷) or 8-CPT-cAMP (20mg/kg) and M1 virus (3 × 10 PFU⁷PFU), successive administration two days.After administration Three put to death nude mice everyday, collect tissue sample (including tumour, the heart, liver, spleen, lung, kidney, brain, muscle), and extract tissue RNA.So Afterwards, the amount of M1 viruses is detected with the method for QRT-PCR.

As a result：

As shown in figure 8, in nude mice by subcutaneous HCT116 tumor models, M1 viruses are in tumor tissues after independent M1 virus treateds Inside there is less M1 viral RNAs.Compared with independent M1 virus treateds group, 8-CPT-cAMP (20mg/kg) combines M1 viruses (3 ×10⁷PFU) quantity of M1 viral RNAs is more than 1000 times or more in the tumour of processing group, and the M1 diseases in other normal structures Malicious RNA does not increase significantly.Illustrate that 8-CPT-cAMP can selectively increase M1 viruses and be replicated in tumor tissues, M1 Virus is enriched in tumor tissues.

2) the viral combined processing of Epac agonists/M1 effectively inhibits tumour growth in mice with tumor body

Material：

M1 viruses, human hepatoma cell strain Hep3B, Human colorectal cancer cells strain HCT116, human pancreas cancer cell strain Capan- 1,4 week old female BAl BIcs/c nude mices

Method：

A) mice with tumor model foundation：By 5 × 10⁶Hep3B, HCT116 or Capan-1 cell are injected into 4 week old BALB/c Nude mice dorsal subcutaneous.

B) intravenously administrable：When Hep3B cell tumour volumes reach about 50mm³When, intravenous injection M1 viruses (3 × 10⁷PFU/ It is secondary)/Epac agonists.OptiPRO^TMThe injection of SFM culture mediums is solvent control group.The length and width of tumour are measured every three days, tumour Volume is according to formula：It is long × wide²/2。

As a result：

After establishing subcutaneous lotus knurl Hep3B, HCT116, Capan-1 (Fig. 9 a, b, c) nude mice model on BALB/c nude mices, Solvent control, M1 viruses, 8-CPT-cAMP, 8-CPT-cAMP joint M1 virus treateds, observation are given in continuous several times intravenous injection Nude mouse tumor volume change situation.Statistics shows to compare with control group or drug alone processing group, and 8-CPT-cAMP combines M1 diseases Malicious processing group significantly inhibits Hep3B/HCT116/Capan-1 mice with tumor tumour growths, and the state of mind is good.Show 8-CPT- CAMP joint M1 virus treated ratios are applied alone M1 viruses, the more efficient inhibition ground tumour growth of 8-CPT-cAMP cAMP groups are applied alone, and And conjunctive use good security.

5 M1 virus preparation methods of embodiment

Material：

African green monkey kidney cell Vero, Chinese hamster ovary cell CHO, human embryonic kidney cells HEK293, DMEM in high glucose culture Base, OptiPRO^TMSFM culture mediums (1 ×), M1 viruses, 100mm Tissue Culture Dish, centrifuge

Method：

Select exponential phase cell, it is outstanding that (containing 10% fetal calf serum, 1% dual anti-) cell is made in DMEM complete mediums Liquid, cell inoculation is in 100mm Tissue Culture Dish.When the degrees of fusion of cell reaches 80%~90%, Vero cells change into OptiPRO^TMSFM culture mediums.The infection of 50 μ l (MOI=0.01) M1 viruses, or processing are added with 500 μM of db-cAMP, when thin There is large area lesion (about 36 hours) in born of the same parents, collect cell conditioned medium.2000~3000RPM centrifuges 5min, and supernatant is carefully sucked out, and mixes Even packing is preserved in -80 DEG C of refrigerators.

As a result：

As shown in Figure 10, in Vero, CHO, in HEK293 cells, individually infection can cause more virus multiple to M1 viruses System significantly increases virus replication after processing is with Epac agonist db-cAMP.Increase to prompt Epac agonists to have The effect of solubilization tumor virus M1 productions.

Claims

1. a kind of pharmaceutical composition for treating tumour, including：

（a）The direct or indirect agonists of Epac, the direct or indirect agonists of Epac are selected from cAMP analogs, adenylate is cyclized One or more combinations in enzyme agonist, phosphodiesterase inhibitors, and

（b）M1 oncolytic virus.

2. pharmaceutical composition according to claim 1, wherein the cAMP analogs are selected from Calcium Dibutyryladenosine Cyclophosph-ate (db-cAMP), 8- (the thio chlorphenyls of 4-) -3 ', 5 '-cyclic adenosine monophosphate（8-CPT-cAMP）, 3 ', 5 '-cyclic phosphorothioic acid glands Glycosides（Rp-cAMPS）, 2'- oxos-(2- ammonia second formoxyl) -3 ', 5 '-cyclic phosphorothioic acid adenosines（Rp-2'-AEC-cAMPS / Rp-2'-EDA-cAMPS）, 8- (6- hexamethylene diamines base) -3 ', 5 '-cyclic phosphorothioic acid adenosines（Rp-8-AHA-cAMPS）, agarose The fixed 8- of gel (6- hexamethylene diamines base) -3 ', 5 '-cyclic phosphorothioic acid adenosines（Rp-8-AHA-cAMPS-Agarose）、8- (2- ethylenediamines base) -3 ', 5 '-cyclic phosphorothioic acid adenosines（Rp-8-AEA-cAMPS）, 8- (2- ethylenediamines base) -2'- oxos-first Base -3 ', 5 '-cyclic adenosine monophosphate（8-AET-2'-O-Me-cAMP）, 8- (6- hexamethylene diamines base) -2'- oxos-methyl -3 ', 5 ' - Cyclic adenosine monophosphate（8-AHA-2'-O-Me-cAMP）, 8- benzylthios -2'- oxos-methyl -3 ', 5 '-cyclic phosphorothioic acid adenosines（Sp- 8-BnT-2'-O-Me-cAMPS / "S-223"）, 8- benzylthios -3 ', 5 '-cyclic phosphorothioic acid adenosines（Sp-8-BnT-cAMPS / "S-220"）, 1-N- oxygroups -3 ', 5 '-cyclic adenosine monophosphate（1-NO-cAMP）, 3 ', 5 '-cyclic adenosine monophosphate（cAMP）、N-6-(2- Aminoethyl) -3 ', 5 '-cyclic phosphorothioic acid adenosines（Sp-6-AE-cAMPS）, 8- (2- [DY-547]-ammonia ethylmercapto group) -3 ', 5 '-rings Adenosine phosphate, chloro- 3 ', the 5 '-cyclic adenosine monophosphate of 8- (6- hexamethylene diamines base) -2-（8-AHA-2-Cl-cAMP）With 8- (6- hexamethylene diamines Base) -3 ', 5 '-cyclic adenosine monophosphate（8-AHA-cAMP）In one or more combinations.

3. pharmaceutical composition according to claim 1, wherein the adenyl cyclase agonist is Forskolin.

4. pharmaceutical composition according to claim 1, wherein the phosphodiesterase inhibitors are selected from 3- isobutyl group -1- first Base xanthine（IBMX）, cilomilast（Cilomilast）, roflumilast（Roflumilast）, digicitrine （Luteolin）, diprophylline（Dyphylline）, rolipram（Rolipram）, sildenafil citrate （Sildenafil Citrate）, Tadalafei（Tadalafil）, three hydrochloride hydrate of Vardenafil（Vardenafil HCl Trihydrate）, pimobendan（Pimobendan）, GSK256066, PF-2545920, Apremilast（Apremilast (CC-10004)）, Cilostazol（Cilostazol）, Milrinone（Milrinone）, avanaphil（Avanafil）, ammonia tea Alkali（Aminophylline）, Dipyridamole（Dipyridamole）, doxofylline（Doxofylline）In Deltarasin It is one or more.

5. pharmaceutical composition according to claim 1, wherein the tumour is solid tumor or blood tumor.

6. pharmaceutical composition according to claim 1, wherein the tumour is selected from liver cancer, colorectal cancer, carcinoma of urinary bladder, mammary gland Cancer, cervical carcinoma, prostate cancer, glioma, melanoma, cancer of pancreas, nasopharyngeal carcinoma, lung cancer, gastric cancer, oophoroma and leukaemia.

7. pharmaceutical composition according to claim 1, wherein the tumour is the tumour insensitive to the oncolytic virus.

8. pharmaceutical composition according to claim 1, wherein described pharmaceutical composition also include pharmaceutically acceptable load Body, and the dosage form of described pharmaceutical composition is selected from freeze-dried powder, injection, tablet, capsule, drops or patch.

9. a kind of drug suit for treating tumour, including：

（a）The direct or indirect agonists of Epac of independent packaging, the direct or indirect agonists of Epac be selected from cAMP analogs, One or more combinations in adenyl cyclase agonist, phosphodiesterase inhibitors, and

（b）The M1 oncolytic virus of independent packaging.

10. drug suit according to claim 9, wherein the cAMP analogs are selected from Calcium Dibutyryladenosine Cyclophosph-ate (db- CAMP), 8- (the thio chlorphenyls of 4-) -3 ', 5 '-cyclic adenosine monophosphate（8-CPT-cAMP）, 3 ', 5 '-cyclic phosphorothioic acid adenosines （Rp-cAMPS）, 2'- oxos-(2- ammonia second formoxyl) -3 ', 5 '-cyclic phosphorothioic acid adenosines（Rp-2'-AEC-cAMPS / Rp-2'-EDA-cAMPS）, 8- (6- hexamethylene diamines base) -3 ', 5 '-cyclic phosphorothioic acid adenosines（Rp-8-AHA-cAMPS）, agarose The fixed 8- of gel (6- hexamethylene diamines base) -3 ', 5 '-cyclic phosphorothioic acid adenosines（Rp-8-AHA-cAMPS-Agarose）、8- (2- ethylenediamines base) -3 ', 5 '-cyclic phosphorothioic acid adenosines（Rp-8-AEA-cAMPS）, 8- (2- ethylenediamines base) -2'- oxos-first Base -3 ', 5 '-cyclic adenosine monophosphate（8-AET-2'-O-Me-cAMP）, 8- (6- hexamethylene diamines base) -2'- oxos-methyl -3 ', 5 ' - Cyclic adenosine monophosphate（8-AHA-2'-O-Me-cAMP）, 8- benzylthios -2'- oxos-methyl -3 ', 5 '-cyclic phosphorothioic acid adenosines（Sp- 8-BnT-2'-O-Me-cAMPS / "S-223"）, 8- benzylthios -3 ', 5 '-cyclic phosphorothioic acid adenosines（Sp-8-BnT-cAMPS / "S-220"）, 1-N- oxygroups -3 ', 5 '-cyclic adenosine monophosphate（1-NO-cAMP）, 3 ', 5 '-cyclic adenosine monophosphate（cAMP）、N-6-(2- Aminoethyl) -3 ', 5 '-cyclic phosphorothioic acid adenosines（Sp-6-AE-cAMPS）, 8- (2- [DY-547]-ammonia ethylmercapto group) -3 ', 5 '-rings Adenosine phosphate, chloro- 3 ', the 5 '-cyclic adenosine monophosphate of 8- (6- hexamethylene diamines base) -2-（8-AHA-2-Cl-cAMP）With 8- (6- hexamethylene diamines Base) -3 ', 5 '-cyclic adenosine monophosphate（8-AHA-cAMP）In one or more combinations.

11. drug suit according to claim 9, wherein the adenyl cyclase agonist is Forskolin.

12. drug suit according to claim 9, wherein the phosphodiesterase inhibitors are selected from 3- isobutyl group -1- first Base xanthine（IBMX）, cilomilast（Cilomilast）, roflumilast（Roflumilast）, digicitrine （Luteolin）, diprophylline（Dyphylline）, rolipram（Rolipram）, sildenafil citrate （Sildenafil Citrate）, Tadalafei（Tadalafil）, three hydrochloride hydrate of Vardenafil（Vardenafil HCl Trihydrate）, pimobendan（Pimobendan）, GSK256066, PF-2545920, Apremilast（Apremilast (CC-10004)）, Cilostazol（Cilostazol）, Milrinone（Milrinone）, avanaphil（Avanafil）, ammonia tea Alkali（Aminophylline）, Dipyridamole（Dipyridamole）, doxofylline（Doxofylline）In Deltarasin It is one or more.

13. drug suit according to claim 9, wherein the tumour is solid tumor or blood tumor.

14. drug suit according to claim 9, wherein the tumour is selected from liver cancer, colorectal cancer, carcinoma of urinary bladder, mammary gland Cancer, cervical carcinoma, prostate cancer, glioma, melanoma, cancer of pancreas, nasopharyngeal carcinoma, lung cancer, gastric cancer, oophoroma and leukaemia.

15. drug suit according to claim 9, wherein the tumour is the tumour insensitive to the oncolytic virus.

16. a kind of method producing M1 oncolytic virus, including：

（a）Cultivate cell；

（b）M1 oncolytic virus is inoculated in the cell of culture；

（c）The direct or indirect agonists of Epac are added into the cell of culture, the direct or indirect agonists of Epac are selected from One or more combinations in cAMP analogs, adenyl cyclase agonist, phosphodiesterase inhibitors；

（d）Cell culture fluid is collected, isolates and purifies to obtain the M1 oncolytic virus.

17. according to the method for claim 16, wherein the cell is selected from：Vero cells, Chinese hamster ovary celI and HEK293 are thin Born of the same parents.

18. according to the method for claim 16, wherein the cAMP analogs select Calcium Dibutyryladenosine Cyclophosph-ate (db-cAMP) , 8- (the thio chlorphenyls of 4-) -3 ', 5 '-cyclic adenosine monophosphate（8-CPT-cAMP）, 3 ', 5 '-cyclic phosphorothioic acid adenosines（Rp- cAMPS）, 2'- oxos-(2- ammonia second formoxyl) -3 ', 5 '-cyclic phosphorothioic acid adenosines（Rp-2'-AEC-cAMPS / Rp-2'- EDA-cAMPS）, 8- (6- hexamethylene diamines base) -3 ', 5 '-cyclic phosphorothioic acid adenosines（Rp-8-AHA-cAMPS）, Ago-Gel it is solid Fixed 8- (6- hexamethylene diamines base) -3 ', 5 '-cyclic phosphorothioic acid adenosines（Rp-8-AHA-cAMPS-Agarose）, 8- (2- second two Amido) -3 ', 5 '-cyclic phosphorothioic acid adenosines（Rp-8-AEA-cAMPS）, 8- (2- ethylenediamines base) -2'- oxos-methyl -3 ', 5 '-cyclic adenosine monophosphate（8-AET-2'-O-Me-cAMP）, 8- (6- hexamethylene diamines base) -2'- oxos-methyl -3 ', 5 '-cycli phosphates Adenosine（8-AHA-2'-O-Me-cAMP）, 8- benzylthios -2'- oxos-methyl -3 ', 5 '-cyclic phosphorothioic acid adenosines（Sp-8-BnT- 2'-O-Me-cAMPS / "S-223"）, 8- benzylthios -3 ', 5 '-cyclic phosphorothioic acid adenosines（Sp-8-BnT-cAMPS / "S- 220"）, 1-N- oxygroups -3 ', 5 '-cyclic adenosine monophosphate（1-NO-cAMP）, 3 ', 5 '-cyclic adenosine monophosphate（cAMP）, N-6- (2- ammonia second Base) -3 ', 5 '-cyclic phosphorothioic acid adenosines（Sp-6-AE-cAMPS）, 8- (2- [DY-547]-ammonia ethylmercapto group) -3 ', 5 '-cycli phosphates Adenosine, chloro- 3 ', the 5 '-cyclic adenosine monophosphate of 8- (6- hexamethylene diamines base) -2-（8-AHA-2-Cl-cAMP）With 8- (6- hexamethylene diamines base)- 3 ', 5 '-cyclic adenosine monophosphate（8-AHA-cAMP）In one or more combinations.

19. according to the method for claim 16, wherein the adenyl cyclase agonist is Forskolin.

20. according to the method for claim 16, wherein the phosphodiesterase inhibitors are selected from 3- isobutyl group -1- methyl yellows Purine（IBMX）, cilomilast（Cilomilast）, roflumilast（Roflumilast）, digicitrine（Luteolin）, two Brontyl（Dyphylline）, rolipram（Rolipram）, sildenafil citrate（Sildenafil Citrate）, he Da Lafei（Tadalafil）, three hydrochloride hydrate of Vardenafil（Vardenafil HCl Trihydrate）, pimobendan （Pimobendan）, GSK256066, PF-2545920, Apremilast（Apremilast (CC-10004)）, Cilostazol （Cilostazol）, Milrinone（Milrinone）, avanaphil（Avanafil）, aminophylline（Aminophylline）, it is double It is phonetic to reach not（Dipyridamole）, doxofylline（Doxofylline）With it is one or more in Deltarasin.

Application of the combination of the direct or indirect agonists of 21.Epac and M1 oncolytic virus in the drug for preparing treatment tumour, Described in the direct or indirect agonists of Epac be selected from cAMP analogs, adenyl cyclase agonist, phosphodiesterase inhibitors In one or more combinations.

22. application according to claim 21, wherein the cAMP analogs are selected from Calcium Dibutyryladenosine Cyclophosph-ate (db- CAMP), 8- (the thio chlorphenyls of 4-) -3 ', 5 '-cyclic adenosine monophosphate（8-CPT-cAMP）, 3 ', 5 '-cyclic phosphorothioic acid adenosines （Rp-cAMPS）, 2'- oxos-(2- ammonia second formoxyl) -3 ', 5 '-cyclic phosphorothioic acid adenosines（Rp-2'-AEC-cAMPS / Rp-2'-EDA-cAMPS）, 8- (6- hexamethylene diamines base) -3 ', 5 '-cyclic phosphorothioic acid adenosines（Rp-8-AHA-cAMPS）, agarose The fixed 8- of gel (6- hexamethylene diamines base) -3 ', 5 '-cyclic phosphorothioic acid adenosines（Rp-8-AHA-cAMPS-Agarose）、8- (2- ethylenediamines base) -3 ', 5 '-cyclic phosphorothioic acid adenosines（Rp-8-AEA-cAMPS）, 8- (2- ethylenediamines base) -2'- oxos-first Base -3 ', 5 '-cyclic adenosine monophosphate（8-AET-2'-O-Me-cAMP）, 8- (6- hexamethylene diamines base) -2'- oxos-methyl -3 ', 5 ' - Cyclic adenosine monophosphate（8-AHA-2'-O-Me-cAMP）, 8- benzylthios -2'- oxos-methyl -3 ', 5 '-cyclic phosphorothioic acid adenosines（Sp- 8-BnT-2'-O-Me-cAMPS / "S-223"）, 8- benzylthios -3 ', 5 '-cyclic phosphorothioic acid adenosines（Sp-8-BnT-cAMPS / "S-220"）, 1-N- oxygroups -3 ', 5 '-cyclic adenosine monophosphate（1-NO-cAMP）, 3 ', 5 '-cyclic adenosine monophosphate（cAMP）、N-6-(2- Aminoethyl) -3 ', 5 '-cyclic phosphorothioic acid adenosines（Sp-6-AE-cAMPS）, 8- (2- [DY-547]-ammonia ethylmercapto group) -3 ', 5 '-rings Adenosine phosphate, chloro- 3 ', the 5 '-cyclic adenosine monophosphate of 8- (6- hexamethylene diamines base) -2-（8-AHA-2-Cl-cAMP）With 8- (6- hexamethylene diamines Base) -3 ', 5 '-cyclic adenosine monophosphate（8-AHA-cAMP）In one or more combinations.

23. application according to claim 21, wherein the adenyl cyclase agonist is selected from Forskolin.

24. application according to claim 21, wherein the phosphodiesterase inhibitors are selected from 3- isobutyl group -1- methyl yellows Purine（IBMX）, cilomilast（Cilomilast）, roflumilast（Roflumilast）, digicitrine（Luteolin）, two Brontyl（Dyphylline）, rolipram（Rolipram）, sildenafil citrate（Sildenafil Citrate）, he Da Lafei（Tadalafil）, three hydrochloride hydrate of Vardenafil（Vardenafil HCl Trihydrate）, pimobendan （Pimobendan）, GSK256066, PF-2545920, Apremilast（Apremilast (CC-10004)）, Cilostazol （Cilostazol）, Milrinone（Milrinone）, avanaphil（Avanafil）, aminophylline（Aminophylline）, it is double It is phonetic to reach not（Dipyridamole）, doxofylline（Doxofylline）With it is one or more in Deltarasin.

25. application according to claim 21, wherein the tumour is solid tumor or blood tumor.

26. application according to claim 21, wherein the tumour be selected from liver cancer, colorectal cancer, carcinoma of urinary bladder, breast cancer, Cervical carcinoma, prostate cancer, glioma, melanoma, cancer of pancreas, nasopharyngeal carcinoma, lung cancer, gastric cancer, oophoroma and leukaemia.

27. application according to claim 21, wherein the tumour is the tumour insensitive to the oncolytic virus.