CN1608131A - Drug for adenocarcinoma of pancreas - Google Patents

Abstract

The invention concerns a medicament to treat a fibrotic disease, wherein the medicament contains a double-stranded ribonucleic acid (dsRNA) that is suitable for inhibiting by RNA interference the expression of a gene that is involved in the formation of extracellular matrix.

Description

The medicine of treatment carcinoma of the pancreas

The present invention relates to treat the medicine and the purposes of carcinoma of the pancreas, and the purposes of producing this medicine.

The gland cancer of carcinoma of the pancreas and pancreas is respectively one of cancer that has the most bad prognosis.The cause of disease for carcinoma of the pancreas is not also known now.Also there is not suitable therapy by now.Except the change of other gene, pancreatic cancer cell often is presented at the sudden change in the K-ras gene.Especially in codon 12,13 and 61, detected sudden change.The K-ras gene is that coding GTP-is in conjunction with the proteic proto-oncogene of K-ras.K-ras is positioned in the tenuigenin side of cytoplasmic membrane, and relevant with acceptor-casein-kinases.The combination activation K-ras of GTP.Hydrolytic rupture in conjunction with the phosphoric acid residue of GTP causes inactivation.The result discharges the GDP of formation, and the recombine of GTP can cause the reactivate of K-ras.Above-mentioned sudden change can cause amino acid whose exchange among the K-ras, thereby causes its lasting activation.The activation of K-ras is gone back activated protein kinase C except that other effect.

Known a kind of method that suppresses expression of target gene in the cell from German patent DE 10100586C1, the few Yeast Nucleic Acid that wherein will have duplex structure is introduced cell.Chain in the duplex structure and target gene complementation.In this method, inhibiting principle is called as RNA and disturbs.

Task of the present invention is the shortcoming that overcomes prior art.Particularly obtain the active drug and the purposes of treatment carcinoma of the pancreas.And, the purposes that obtains to produce this medicine.

This task solves by the feature in the claim 1,19 and 20.Accessory Right requires the feature in 2 to 18 and 21 to 38 can draw embodiment preferred.

According to the present invention, contain the medicine that is fit to the double-stranded ribose thuja acid (dsRNA) by RNA conflicting mode inhibition K-ras genetic expression and be used for the treatment of carcinoma of the pancreas, particularly human pancreas cancer.Not that all Nucleotide among the dsRNA all must be shown as typical Wo Sen-Ke Like (Watson-Crick) base pair.Especially, one, non-complementary base pair does not almost damage effect.The base logarithm of maximum possible also is the few nucleotide in the contained short chain among the dsRNA.

This medicine contains the dsRNA that is enough to suppress K-ras genetic expression dosage in the carcinoma of the pancreas.Also this medicinal design can be become the summation of several pharmaceutical units to contain the such mode of q.s jointly.Q.s depends on the mode of administration.In order to determine needed amount, can give dsRNA with amount or the dosage that increases gradually.Then whether the tissue sample that obtains from carcinoma of the pancreas by the currently known methods check K-ras expression of gene takes place when this amount and suppresses to detect.These methods for example may comprise molecular biology, biological chemistry or immunologic method.

Make the people be in surprise,, shown the propagation that can suppress pancreatic cancer cell, and can reduce the quantity of tumour cell alive by with the dsRNA treatment that can optionally suppress K-ras genetic expression.Amazing is that the propagation behavior of non-malignant cell is not subjected to the influence of this therapy to a great extent.This medicine can increase the apoptosis rate in the pancreatic cancer cell.In vivo, use such medicine may suppress the growth of pancreatic neoplasm effectively.

Can influence the lasting activation of K-ras in the such mode of sudden change K-ras gene.It is effective especially in the growth that suppresses carcinoma of the pancreas to suppress this expression of gene by medicine according to the present invention.The K-ras gene can suddenly change in codon 12,13 or 61.By in the K-ras gene that suddenlys change, codon 12 can encode arginine, Serine, L-Ala, Xie Ansuan, halfcystine or aspartic acid, perhaps codon 13 aspartic acid of can encoding, perhaps codon 61 can encoding histidine or leucines.In wild-type K-ras gene, codon 12 and codon 13 each glycine of all encoding, codon 61 coding L-glutamic acid.

Preferably, the chain S1 of dsRNA has a zone, constitutes by being less than 25 continuous nucleotides in particular, and it is to small part and K-ras gene complementation.Such dsRNA is very suitable for suppressing the K-ras expression of gene especially." K-ras gene " should be understood that to refer to the DNA chain of the double-stranded DNA of encoded K-ras in tumour cell, its with comprise all by transcriptional domain, as the DNA chain complementation of transcribing template.Therefore K-ras gene sense strand normally.Like this S1 chain can with rna transcription this or its processed products, for example the mRNA complementation that in K-ras expression of gene process, forms.

Complementary dsRNA district may have 19 to 24, and is preferred 20 to 24, preferred especially 21 to 23, especially preferred 22 or 23 Nucleotide.DsRNA efficient in suppressing the K-ras gene with this structure is high especially.The S1 chain of dsRNA may have and is less than 30, preferably is less than 25, and is preferred especially 21 to 24, especially preferred 23 Nucleotide.The quantity of these Nucleotide also is the base logarithm of maximum possible in dsRNA.This dsRNA is stable especially in cell.

The verified at least one end as dsRNA has by 1 to 4, particularly advantageous during the strand overhang that is made of 2 or 3 Nucleotide especially.With do not have the dsRNA of strand overhang to compare at least one end, this dsRNA has shown superior effect in suppressing the K-ras expression of gene.Here, an end is to have 5 '-and the dsRNA district of 3 '-chain end.Only the dsRNA that is made of the S1 chain correspondingly has ring texture and an end only.The dsRNA that is made of S1 chain and S2 chain has two ends.In all cases, an end is formed by the chain end of chain S1, and the other end is formed by the chain end of chain S2.

The strand overhang is preferably placed at 3 ' of S1 chain-end.This position of strand overhang causes the further raising of drug efficiency.In an example, dsRNA only at one end particularly shows the strand overhang at an end that is positioned at S1 chain 3 ' end.The other end is a flush end in the dsRNA that shows two ends,, lacks overhang that is.Verified such dsRNA is stable especially in various cell culture mediums and in blood and serum.

Preferably, dsRNA also has the S2 chain except having the S1 chain, that is, it is made of two strands.When S1 chain (antisense strand) is that 23 Nucleotide are long, the S2 chain is that 21 Nucleotide are long, and 3 '-end of S1 chain when having the strand overhang that constitutes by two Nucleotide this medicine effective especially.Here the dsRNA end that is positioned at S1 chain 5 '-end is a flush end.The S1 chain can with the elementary of K-ras gene or this complementation of the rna transcription of having processed.Preferably, dsRNA is by S2 chain with SEQ ID NO:1 sequence and the S1 chain with SEQ ID NO:2 sequence, or by S2 chain with SEQ ID NO:3 sequence and S1 chain with SEQ ID NO:4 sequence, or constitute by S2 chain with SEQ ID NO:5 sequence and S1 chain with SEQ ID NO:6 sequence, as shown in appended sequence table.This dsRNA is effective especially in suppressing the K-ras expression of gene.

DsRNA can be present in the medicine in solution, particularly in the damping fluid or normal saline solution that can tolerate on the physiology, perhaps centered on by micellar structure, preferably by liposome, capsid, class capsomere, or polymer nano capsule or microcapsule center on, and perhaps are incorporated on polymer nano capsule or the micro-capsule.

The damping fluid that tolerates on the physiology may be a phosphoric acid buffers saline solution.Micellar structure, capsid, class capsomere, or polymer nano capsule or microcapsule can be convenient to the picked-up of dsRNA in tumour cell.Polymer nano capsule or microcapsule are by at least a biodegradable polymkeric substance, and for example poly-butyl cyanoacrylate constitutes.Polymer nano capsule or microcapsule can be transported in vivo and discharge and comprise within it or be incorporated into dsRNA on it.

Medicine can be shown as be suitable for sucking, oral, infusion or injection, particularly be suitable for the preparation of infusion in intravenously, intraperitoneal or the tumour or injection.Be suitable for sucking, the preparation of infusion or injection is made of the damping fluid, particularly phosphoric acid buffers saline solution that can tolerate on preferred normal saline solution or the physiology the most simply the solvent that can tolerate on dsRNA and the physiology.Make the people be in surprise, shown that dsRNA needn't be wrapped in the specific carrier, the dsRNA of dissolving and administration also can be taken in by tumour cell in such solvent or such buffer reagent simply, and inhibition K-ras expression of gene.

Preferably, medicine exists with at least one dose unit, and it is the amount of the dsRNA of per kilogram of body weight 5mg every day, 2.5mg, 200 μ g, 100 μ g, 50 μ gh and the suitableeest 25 μ g dsRNA that this dose unit contains the maximal dose that can arrange with the preferred sequence of rising progressively.Verified in addition when this dosage dsRNA also demonstrate the unusual effect that suppresses K-ras genetic expression.Dose unit can be designed to single-dose or take in dosage every day.In this case, comprise whole per daily doses in the single dosage unit.If dose unit is designed to the dosage of the every day of administration several times or absorption, in order to obtain the total dose of every day, dsRNA amount contained in each dosage will correspondingly diminish so.Also dose unit can be designed to for example amount of single-dose or absorption in several days, the result discharged dsRNA in several days.So dose unit contains corresponding a plurality of every day of dosage.

In addition, according to the present invention, purpose is to be fit to be used for by the double stranded RNA that RNA interferential mode suppresses K-ras genetic expression the medicine of production for treating carcinoma of the pancreas.In addition, according to the present invention, purpose is that the double stranded RNA that will be fit to suppress by RNA interferential mode K-ras genetic expression is used for the treatment of carcinoma of the pancreas.

According to the present invention, about the see before commentary of face of the advantageous embodiments of purposes.

Below, will the present invention be described exemplaryly with figure.With abbreviation " M " replacement " mol/l " indicated concentration.The apoptosis rate (percentage) that in Fig. 1, shows human pancreatic cancer cell YAP C depend on the first sequence complementary dsRNA transfection of human K-ras gene after the cultivation time, in Fig. 2, show quantity, in Fig. 3, be presented at the volume of human pancreas's gland cancer of subcutaneous implantation in the NMRI-mouse with viable cell after the dsRNA transfection.

The double-stranded oligoribonucleotide that uses is shown as following sequence, is called SEQID NO:1 to SEQ ID NO:8 in sequence table:

KRAS1, and the human K-ras gene order complementation that in codon 12, shows first point mutation in the YAP C cell:

S2：5’-agu?ugg?agc?ugu?ugg?cgu?agg-3’(SEQ?ID?NO：1)

S1：3’-ca?uca?acc?ucg?aca?acc?gca?ucc-5’(SEQ?ID?NO：2)

KRAS1 ', and the human K-ras gene order complementation that in codon 12, shows first point mutation in human pancreas's gland cancer of NMRI mouse subcutaneous transplanting:

S2：5’-agu?ugg?agc?uga?ugg?cgu?agg-3’(SEQ?ID?NO：3)

S1：3’-ca?uca?acc?ucg?acu?acc?gca?ucc-5’(SEQ?ID?NO：4)

KRAS2, and wild-type sequence complementation from the human K-ras gene:

S2：5’-agu?ugg?agc?ugg?ugg?cgu?agg-3’(SEQ?ID?NO：5)

S1：3’-ca?uca?acc?ucg?acc?acc?gca?ucc-5’(SEQ?ID?NO：6)

NEO, and sequence complementation from neomycin resistance gene:

S2：5’-c?aag?gau?gag?gau?cgu?uuc?gca-3’(SEQ?ID?NO：7)

S1：3’-ucu?guc?cua?cuc?cua?gca?aag?cg-5’(SEQ?ID?NO：8)

At 37 ℃, 5%CO ₂Under the controlled condition, at the RPMI1640 substratum (Bio-chrom that contains 10% foetal calf serum (FCS) and 100 μ g/ml penicillin/streptomycin, Berlin) cultivation can be from the human pancreatic cancer cell YAP C cell of German microorganism and the acquisition of cell culture preservation center, Braunschweig (No.ACC 382) in.

(Invitrogen Karlsruhe) carries out transfection with oligofectamine in 6 orifice plates.At 150,000 cells of each hole middle berth.Scheme according to the Invitrogen suggestion is carried out the transfection (data that relate to a hole in 6 orifice plates) of double-stranded oligoribonucleotide with oligofectamine: dilute the double-stranded oligoribonucleotide (0.1 to 10 μ M) of 10 μ l with the non-additive cell culture medium of 175 μ l.Dilute 3 μ loligofectamine with the non-additive cell culture medium of 12 μ l, at room temperature cultivated 10 minutes.The oligofectamine of dilution in this way is added to dilutes in the good double-stranded oligoribonucleotide, mix, at room temperature cultivated 20 minutes.In the meantime, with non-additive cell culture medium flushing once, add the fresh cell culture medium of 800 μ l with transfected cell.Then in each hole, add the oligofectamine-dsRNA mixture that 200 μ l were described, make that final infection volume is 1000 μ l.

This final concentration that causes double-stranded oligoribonucleotide is 1-100nM.Cultivated the transfection analyte 4 hours at 37 ℃.After this, in each hole, add the cell culture medium that 500 μ l contain 30%FCS, make that the final concentration of FCS is 10%.Then cultivated this analyte 24 to 120 hours at 37 ℃.

In order to measure apoptosis rate, cultivate the back and collect supernatant liquor, with phosphoric acid buffers saline solution (PBS) flushing cell, use trypsinase to separate, under 100g centrifugal 10 minutes.Abandoning supernatant is deposited in the hypotonic iodate third ingot solution and cultivated 30 minutes in 4 ℃ of dark.(BD GmbH Heidelberg) analyzes by the method for flow cytometry with fluoroscopic assist cell sorter FACSCalibur.

Fig. 1 has shown the apoptosis rate (pressing percentage) of human pancreatic cancer cell YAP C, when its incubation time after to the KRAS1 dsRNA transfection that increases concentration gradually has dependence.Can see from this, KRAS1 can be in human pancreatic cancer cell the apoptosis that relies on of induced concentration.Apoptosis rate increases along with the increase of incubation time.And do not have processed YAP C cell (contrast) and double-stranded oligoribonucleotide of no use to carry out described transfection method cells transfected (pseudo-transfection or false transfection) after 120 hours cultivation, also only shown 5% maximum apoptosis rate, be increased to 24% and cultivated the back apoptosis rate at 120 hours with the 100nMKRAS1 cells transfected.In YAP C cell, induce the apoptosis of same effect with K-ras wild-type complementary KRAS2 dsRNA.

In order to measure transfection, spread 50,000 YAP C cells in each hole in 6 orifice plates respectively and according to method transfection described above to the propagation and the influence of viable cell quantity.After cultivating in 24 to 120 hours, get rid of dyeing, measure the quantity of viable cell by counting in the Neubauer nucleonics with trypan blue.The result is presented among Fig. 2.The propagation of YAP C cell is subjected to the inhibition of KRAS1 and depends on the concentration of KRAS1.Only using the KRAS1 of 1nM that the quantity of viable cell is reduced statistically significantly (compares with untreated contrast after 120 hours, p=0.001).

Under 100nM concentration, cause the minimizing of viable cell quantity with K-ras wild-type complementary KRAS2 dsRNA transfection.In their propagation behavior, shown variation by non-pernicious human skin fibroblast with KRAS1 or KRAS2 transfection.

For testing in the body, with human pancreas's adenocarcinoma tissue fragment subcutaneous transplantation of diameter 2-3mm to the NMRI mouse (Harlan Winkelmann GmbH, Borchen) in.Long after the 6-7mm size in tumour, with being dissolved in the KRAS1 ' of physiological salt solution or the dosage that NEO presses per kilogram of body weight 200 μ g, carry out peritoneal injection every day.Injecting normal saline solution in contrast.Measure the size of tumour every day with vernier callipers or standard form.Fig. 3 shows the tumor size of being surveyed with the standard error of mean value ± mean value, and unit is mm ³, depend on begin with injection for curing under the peritonaeum Measuring Time that fate represents (fate, i.p.).Can see that from this dsRNA with the K-ras gene complementation can suppress growth of tumor.Suppress growth of tumor by the dsRNA that uses down with dosage peritonaeum every day of 200 μ g/kg with the K-ras gene complementation, make after 24 days the treatment, tumor size only be seen in control group gross tumor volume 62%.

Sequence table

<110>Ribopharma?AG

＜120〉medicine of treatment carcinoma of the pancreas

<130>422271EH

<140>

<141>

<160>8

<170>Patent?In?Ver.2.1

<210>1

<211>21

<212>RNA

＜213〉people

<400>1

aguuggagcu?guuggcguag?g 21

<210>2

<211>23

<212>RNA

＜213〉people

<400>2

ccuacgccaa?cagcuccaac?uac 23

<210>3

<211>21

<212>RNA

＜213〉people

<400>3

aguuggagcu?gauggcguag?g 21

<210>4

<211>23

<212>RNA

＜213〉people

<400>4

ccuacgccau?cagcuccaac?uac 23

<210>5

<211>21

<212>RNA

＜213〉people

<400>5

aguuggagcu?gguggcguag?g 21

<210>6

<211>23

<212>RNA

＜213〉people

<400>6

ccuacgccac?cagcuccaac?uac 23

<210>7

<211>22

<212>RNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: with the sense strand of neomycin resistance gene sequence complementary dsRNA

<400>7

caaggaugag?gaucguuucg?ca 23

<210>8

<211>23

<212>RNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: with the antisense strand of neomycin resistance gene sequence complementary dsRNA

<400>8

gcgaaacgau?ccucauccug?ucu 23

Claims (38)

1. treat the medicine of carcinoma of the pancreas, wherein this medicine contains the double stranded RNA (dsRNA) that is fit to suppress by RNA interferential mode K-ras genetic expression.

2. according to the medicine of claim 1, the K-ras that wherein suddenlys change causes it to cause the continuous activation of K-ras.

3. according to the medicine of claim 1 or 2, wherein the K-ras gene suddenlys change in codon 12,13 or 61.

4. according to the medicine of one of front claim, wherein codon 12 coding arginine, Serine, L-Ala, Xie Ansuan, halfcystine or aspartic acid in the K-ras gene, codon 13 coding aspartic acid, perhaps codon 61 encoding histidines or leucines.

5. according to the medicine of one of front claim, wherein the S1 chain of dsRNA has to the zone of small part and K-ras gene complementation, should constitute by being less than 25 continuous nucleotides in the zone especially.

6. according to the medicine of one of front claim, wherein complementary region has 19-24, preferred 20-24, preferred especially 21-23, especially preferred 22 or 23 Nucleotide.

7. according to the medicine of one of front claim, wherein the S1 chain has and is less than 30, preferably is less than 25, preferred especially 21-24, especially preferred 23 Nucleotide.

8. according to the medicine of one of front claim, wherein the end of dsRNA has by 1-4 at least, especially by 2 or 3 strand overhangs that Nucleotide constitutes.

9. medicine according to Claim 8, wherein the strand overhang is positioned at 3 ' of S1 chain-end.

10. according to the medicine of one of front claim, wherein dsRNA only at one end particularly has the strand overhang at the end that is positioned at S1 chain 3 '-end.

11. according to the medicine of one of front claim, wherein dsRNA also has the S2 chain except that having the S1 chain.

12. according to the medicine of claim 11, wherein the S1 chain is that 23 Nucleotide are long, the S2 chain is that 21 Nucleotide are long, and 3 '-end of S1 chain has the strand overhang that is made of two Nucleotide, and the dsRNA end that is positioned at S1 chain 5 '-end is a flush end.

13. according to the medicine of one of front claim, wherein S1 chain and K-ras gene elementary or this complementation of rna transcription of having processed.

14. medicine according to one of front claim, wherein dsRNA is by S2 chain with SEQ IDNO:1 sequence and the S1 chain with SEQ ID NO:2 sequence, or by S2 chain with SEQ IDNO:3 sequence and S1 chain with SEQ ID NO:4 sequence, or constitute by S2 chain with SEQ IDNO:5 sequence and S1 chain with SEQ ID NO:6 sequence, as shown in appended sequence table.

15. medicine according to one of front claim, dsRNA in its Chinese traditional medicine exists in solution, particularly in the damping fluid or normal saline solution that can tolerate on the physiology, centered on by micellar structure, preferably centered on, perhaps be incorporated on polymer nano capsule or the microcapsule by liposome, capsid, class capsomere or polymer nano capsule or microcapsule.

16. according to the medicine of one of front claim, its Chinese traditional medicine is shown as and be fit to sucks, oral, infusion or injection, is fit to the preparation of infusion in intravenously, intraperitoneal or the tumour or injection especially.

17. according to the medicine of claim 16, wherein said preparation, particularly only by the solvent that can tolerate on dsRNA and the physiology, the damping fluid, particularly phosphoric acid buffers saline solution that can tolerate on preferred normal saline solution or the physiology constitute.

18. medicine according to one of front claim, its Chinese traditional medicine is so that a few dose unit exists, and it is every day per kilogram of body weight 5mg, 2.5mg, 200 μ g, 100 μ g, 50 μ g and the dsRNA of the suitableeest 25 μ g that this dose unit contains the maximal dose that can arrange with the preferred sequence of rising progressively.

19. be fit to suppress the purposes of double stranded RNA (dsRNA) in the medicine of production for treating carcinoma of the pancreas of K-ras genetic expression by the RNA conflicting mode.

20. be fit to suppress the purposes of double stranded RNA (dsRNA) in the treatment carcinoma of the pancreas of K-ras genetic expression by the RNA conflicting mode.

21., wherein make the K-ras transgenation cause it to cause the lasting activation of K-ras according to the purposes of one of claim 19 or 20.

22. according to the purposes of one of claim 19-21, wherein the K-ras gene suddenlys change in codon 12,13 or 61.

23. according to the purposes of one of claim 19-22, wherein codon 12 coding arginine, Serine, Xie Ansuan, halfcystine or aspartic acid in the K-ras gene, codon 13 coding aspartic acid, perhaps codon 61 encoding histidines or leucines.

24. according to the purposes of one of claim 19-23, wherein the S1 chain of dsRNA has at least in part the zone with the K-ras gene complementation, should constitute by being less than 25 continuous nucleotides in the zone especially.

25. according to the purposes of one of claim 19-24, wherein the complementary zone has 19-24, preferred 20-24, preferred especially 21-23, especially preferred 22 or 23 Nucleotide.

26. according to the purposes of one of claim 19-25, wherein the S1 chain has and is less than 3, preferably is less than 25, preferred especially 21-24, especially preferred 23 Nucleotide.

27., wherein have by 1-4, especially by 2 or 3 strand overhangs that Nucleotide constitutes at the end of dsRNA at least according to the purposes of one of claim 19-26.

28. according to the purposes of claim 27, wherein the strand overhang is positioned at 3 ' of S1 chain-end.

29. according to the purposes of one of claim 19-28, wherein dsRNA only at one end has the strand overhang at the end that is positioned at S1 chain 3 '-end especially.

30. according to the purposes of one of claim 19-29, wherein said dsRNA except having the S1 chain, also described S2 chain.

31. according to the purposes of claim 30, wherein said S1 chain is that 23 Nucleotide are long, the S2 chain is that 21 Nucleotide are long, and 3 '-end of S1 chain has the strand overhang that is made of two Nucleotide, and the dsRNA end that is positioned at S1 chain 5 '-end is a flush end.

32. according to the purposes of one of claim 19-31, wherein said S1 chain and K-ras gene elementary or this complementation of rna transcription of having processed.

33. purposes according to one of claim 19-32, wherein said dsRNA is by S2 chain with SEQ ID NO:1 sequence and the S1 chain with SEQ ID NO:2 sequence, or by S2 chain with SEQ ID NO:3 sequence and S1 chain with SEQ ID NO:4 sequence, or constitute by S2 chain with SEQ ID NO:5 sequence and S1 chain with SEQ ID NO:6 sequence, as shown in appended sequence table.

34. purposes according to one of claim 19-33, wherein said dsRNA is present in solution, particularly in the damping fluid or normal saline solution that can tolerate on the physiology, centered on by micellar structure, preferably centered on, perhaps be incorporated on polymer nano capsule or the microcapsule by liposome, capsid, class capsomere or polymer nano capsule or microcapsule.

35. according to the purposes of one of claim 19-34, wherein dsRNA be present in be fit to suck, oral, infusion or injection, particularly be fit in the preparation of infusion in intravenously, intraperitoneal or the tumour or injection.

36. according to the purposes of claim 35, wherein said preparation, especially only by the solvent that can tolerate on dsRNA and the physiology, the damping fluid that tolerates on preferred normal saline solution or the physiology, phosphoric acid buffers saline solution constitutes especially.

37. according to the purposes of one of claim 19-36, wherein said dsRNA is by oral, suction, infusion or injection, especially mode administration by intravenously, peritonaeum infusion or injection down or in the tumour.

38. according to the purposes of one of claim 19-37, wherein dsRNA is by the preferred sequence of rising progressively, per kilogram of body weight maximal dose 5mg, 2.5mg, 200 μ g, 100 μ g, 50 μ g and dose,optimum 25 μ g use with every day.

Images