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

Drugs to treat pancreatic cancer

The present invention relates to the medicine and application for treating pancreatic cancer, and the application of producing the medicine.

Pancreatic cancer and adenocarcinoma of the pancreas, respectively, are among the cancers with the worst prognosis. The cause of pancreatic cancer is not yet known. To date there is no adequate therapy. Pancreatic cancer cells often show mutations in the K-ras gene, among other genetic changes. Mutations have been detected in codons 12, 13 and 61 in particular. The K-ras gene is a proto-oncogene encoding the GTP-binding K-ras protein. K-ras is localized on the cytoplasmic side of the plasma membrane and is associated with receptor-casein-kinase. Binding of GTP activates K-ras. Hydrolytic cleavage of phosphate residues that have bound GTP results in inactivation. As a result of the release of formed GDP, recombination of GTP can lead to reactivation of K-ras. The aforementioned mutations can lead to an amino acid exchange in K-ras, thus resulting in its persistent activation. Activation of K-ras activates protein kinase C, among other things.

A method for inhibiting the expression of a target gene in a cell is known from German patent DE10100586C1, wherein an oligoribonucleic acid having a double-stranded structure is introduced into the cell. One strand of the double-stranded structure is complementary to the target gene. In this method, the principle of inhibition is called RNA interference.

The object of the present invention is to overcome the disadvantages of the prior art. In particular, access to effective drugs and uses for the treatment of pancreatic cancer. Moreover, use is obtained for the production of this drug.

This object is solved by the features of claims 1 , 19 and 20 . Preferred embodiments emerge from the features in claims 2 to 18 and 21 to 38 .

According to the present invention, the medicine containing double-stranded nucleotide (dsRNA) suitable for suppressing the expression of K-ras gene by means of RNA interference is used for treating pancreatic cancer, especially human pancreatic cancer. Not all nucleotides in a dsRNA have to appear as typical Watson-Crick base pairs. In particular, single, non-complementary base pairs hardly impair the effect at all. The maximum possible number of base pairs is also the number of nucleotides in the shortest strand contained in a dsRNA.

The drug contains dsRNA at a dose sufficient to suppress K-ras gene expression in pancreatic cancer. The drug can also be designed in such a way that the sum of several drug units together contains a sufficient amount. Sufficient amounts depend on the mode of administration. To determine the required amount, the dsRNA can be administered in increasing amounts or doses. Tissue samples obtained from pancreatic cancer were then examined by known methods to detect whether suppression of K-ras gene expression occurred at this amount. These methods, for example, may include methods of molecular biology, biochemistry or immunology.

Surprisingly, treatment with dsRNA that selectively inhibits K-ras gene expression has been shown to inhibit the proliferation of pancreatic cancer cells and reduce the number of viable tumor cells. Surprisingly, the proliferative behavior of non-malignant cells was largely unaffected by the therapy. The drug was able to increase the rate of apoptosis in pancreatic cancer cells. In vivo, the use of such drugs may effectively inhibit the growth of pancreatic tumors.

The persistent activation of K-ras can be affected in such a way as to mutate the K-ras gene. Inhibition of the expression of this gene by the medicament according to the invention is particularly effective in inhibiting the growth of pancreatic cancer. The K-ras gene can be mutated in codons 12, 13 or 61. In the mutated K-ras gene, codon 12 can encode arginine, serine, alanine, valine, cysteine or aspartic acid, or codon 13 can encode aspartic acid, Alternatively codon 61 could encode histidine or leucine. In the wild-type K-ras gene, codons 12 and 13 each encode glycine, and codon 61 encodes glutamic acid.

Preferably, one strand S1 of the dsRNA has a region, in particular consisting of less than 25 consecutive nucleotides, which is at least partially complementary to the K-ras gene. Such dsRNAs are particularly well suited for inhibiting the expression of the K-ras gene. "K-ras gene" should be understood as referring to the DNA strand of double-stranded DNA encoding K-ras in tumor cells, which is complementary to the DNA strand including all transcribed regions as transcription templates. Therefore, the K-ras gene is usually a sense strand. In this way, the S1 strand can be complementary to the RNA transcript or its processed product, such as the mRNA formed during the expression of the K-ras gene.

The complementary dsRNA region may have 19 to 24, preferably 20 to 24, particularly preferably 21 to 23, especially preferably 22 or 23 nucleotides. dsRNAs with this structure are particularly efficient at inhibiting the K-ras gene. The S1 strand of the dsRNA may have less than 30, preferably less than 25, particularly preferably 21 to 24, especially preferably 23 nucleotides. The number of these nucleotides is also the largest possible number of base pairs in a dsRNA. This dsRNA is particularly stable inside the cell.

It has proven to be particularly advantageous when at least one end of the dsRNA has a single-stranded overhang consisting of 1 to 4, in particular 2 or 3 nucleotides. This dsRNA exhibited a superior effect in inhibiting the expression of K-ras gene compared to a dsRNA without a single-stranded overhang at at least one end. Here, one end is a dsRNA region where 5'- and 3'-strand ends exist. A dsRNA consisting only of the S1 strand accordingly has a circular structure and only one end. A dsRNA composed of an S1 strand and an S2 strand has both ends. In each case, one end is formed by a chain end of chain S1 and the other end is formed by a chain end of chain S2.

The single-stranded overhang is preferably located at the 3'-end of the S1 strand. This location of the single-stranded overhang leads to a further increase in drug efficiency. In one example, the dsRNA exhibits a single-stranded overhang at only one end, particularly the end at the 3' end of the S1 strand. In dsRNAs showing both ends, the other end is blunt, ie, lacks an overhang. Such dsRNAs have been shown to be particularly stable in a wide variety of cell culture media as well as in blood and serum.

Preferably, the dsRNA has an S2 strand in addition to an S1 strand, ie it consists of two single strands. The drug when the S1 strand (antisense strand) is 23 nucleotides long, the S2 strand is 21 nucleotides long, and the 3'-end of the S1 strand has a single-stranded overhang consisting of two nucleotides Very effective. The dsRNA end located at the 5'-end of the S1 strand is blunt here. The S1 strand is able to complement primary or processed RNA transcripts of the K-ras gene. Preferably, the dsRNA consists of an S2 strand having a SEQ ID NO:1 sequence and an S1 strand having a SEQ ID NO:2 sequence, or an S2 strand having a SEQ ID NO:3 sequence and an S1 strand having a SEQ ID NO:4 sequence , or consist of an S2 strand having the sequence of SEQ ID NO: 5 and an S1 strand having the sequence of SEQ ID NO: 6, as shown in the attached sequence listing. This dsRNA is particularly effective in inhibiting the expression of the K-ras gene.

The dsRNA can be present in the drug in solution, especially in a physiologically tolerable buffer or saline solution, or surrounded by micellar structures, preferably liposomes, capsids, capsids, or polymeric Surrounded by nanocapsules or microcapsules, or bound to polymer nanocapsules or microcapsules.

A physiologically tolerated buffer may be phosphate-buffered saline. Micellar structures, capsids, capsids, or polymeric nanocapsules or microcapsules can facilitate the uptake of dsRNA in tumor cells. Polymeric nanocapsules or microcapsules are composed of at least one biodegradable polymer, such as polybutylcyanoacrylate. Polymeric nanocapsules or microcapsules are capable of transporting and releasing dsRNA contained therein or bound thereto in vivo.

The medicament can be presented as a formulation suitable for inhalation, oral administration, infusion or injection, especially for intravenous, intraperitoneal, or intratumoral infusion or injection. Formulations suitable for inhalation, infusion or injection consist most simply of dsRNA and a physiologically tolerable solvent, preferably physiological saline solution or a physiologically tolerable buffer, especially phosphate-buffered saline. Surprisingly, it has been shown that dsRNA need not be encapsulated in a specific carrier, dsRNA simply dissolved and administered in such a solvent or such a buffer can also be taken up by tumor cells and inhibit the K-ras gene expression.

Preferably, the medicament is present in at least one dosage unit containing an amount of dsRNA that can be arranged in ascending order of preference with a maximum dose of 5 mg, 2.5 mg, 200 μg, 100 μg, 50 μg dsRNA per kg body weight per day and optimally 25 μg dsRNA. It has been demonstrated that even at this dose dsRNA shows a significant effect of inhibiting K-ras gene expression. Dosage units can be formulated for single daily administration or ingestion. In such cases, the entire daily dose will be contained in a single dosage unit. If the dosage unit is designed to be administered or ingested several times per day, the amount of dsRNA contained in each dose will be correspondingly smaller in order to obtain the total daily dose. Dosage units can also be designed such that a single administration or intake over several days results in release of the dsRNA over several days. Dosage unit thus contains a corresponding multiple of the daily dose.

In addition, according to the present invention, the purpose is to use the double-stranded ribonucleic acid suitable for suppressing K-ras gene expression by means of RNA interference for the production of drugs for treating pancreatic cancer. In addition, according to the present invention, the purpose is to use the double-stranded ribonucleic acid suitable for inhibiting the expression of K-ras gene by means of RNA interference for the treatment of pancreatic cancer.

For advantageous embodiments of the use according to the invention see the previous comments.

Hereinafter, the present invention will be exemplarily explained by means of figures. Concentrations are indicated by the abbreviation "M" instead of "mol/l". Show in Fig. 1 that the apoptotic rate (percentage) of human pancreatic cancer cell YAP C is dependent on the cultivation time after the dsRNA transfection complementary to the first sequence of human K-ras gene, in Fig. The number of cells, volume of human pancreatic adenocarcinoma implanted subcutaneously in NMRI-mice is shown in Figure 3 .

The double-stranded oligoribonucleotides used are shown as the following sequences, referred to as SEQ ID NO: 1 to SEQ ID NO: 8 in the Sequence Listing:

KRAS1, complementary to the sequence of the human K-ras gene showing the first point mutation in codon 12 in YAP C cells:

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', complementary to the human K-ras gene sequence showing the first point mutation in codon 12 in human pancreatic adenocarcinoma transplanted subcutaneously in NMRI mice:

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, complementary to the wild-type sequence 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, complementary to the sequence from the 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)

Cultured in RPMI1640 medium (Bio-chrom, Berlin) containing 10% fetal calf serum (FCS) and 100 μg/ml penicillin/streptomycin (Bio-chrom, Berlin) at 37 °C under constant conditions of 5% _CO2 . Human pancreatic cancer cell line YAP C cells obtained from the Culture Collection, Braunschweig (No. ACC 382).

Transfections were performed with oligofectamine (Invitrogen, Karlsruhe) in 6-well plates. 150,000 cells were plated in each well. Transfection of double-stranded oligoribonucleotides with oligofectamine according to the protocol suggested by Invitrogen (involving the data of one well in a 6-well plate): Dilute 10 μl of double-stranded oligoribonucleotides (0.1 to 10 μM). Dilute 3 µl of loligofectamine with 12 µl of supplement-free cell culture medium and incubate at room temperature for 10 min. Add the oligofectamine diluted in this way to the already diluted double-stranded oligoribonucleotides, mix, and incubate at room temperature for 20 minutes. During this period, the cells to be transfected were rinsed once with additive-free cell culture medium and 800 μl of fresh cell culture medium was added. 200 μl of the described oligofectamine-dsRNA mixture was then added to each well, resulting in a final infection volume of 1000 μl.

This results in a final concentration of double-stranded oligoribonucleotides of 1-100 nM. Transfection analytes were incubated for 4 hours at 37°C. After that, 500 µl of cell culture medium containing 30% FCS was added to each well so that the final concentration of FCS was 10%. The analytes were then incubated at 37°C for 24 to 120 hours.

To measure the apoptosis rate, supernatants were collected after incubation, cells were washed with phosphate-buffered saline (PBS), detached using trypsin, and centrifuged at 100 g for 10 minutes. The supernatant was discarded, and the pellet was incubated in hypotonic propidium iodide solution for 30 minutes at 4°C in the dark. Analysis was performed by flow cytometry using a fluorescence-assisted cell sorter FACSCalibur (BD GmbH, Heidelberg).

Figure 1 shows the apoptotic rate (in percentage) of human pancreatic cancer cells YAP C, which is dependent on the culture time after transfection with increasing concentrations of KRAS1 dsRNA. From this, it can be seen that KRAS1 is able to induce concentration-dependent apoptosis in human pancreatic cancer cells. The apoptosis rate increased with the increase of culture time. And the YAP C cell (control) that has not been processed and do not carry out described transfection method transfection cell (sham transfection or false transfection) with double-stranded oligoribonucleotide after cultivating for 120 hours, also only showed a maximal apoptotic rate of 5%, whereas cells transfected with 100 nM KRAS1 increased the apoptotic rate to 24% after 120 h of culture. KRAS2 dsRNA complementary to K-ras wild type induced apoptosis to the same effect in YAP C cells.

To determine the effect of transfection on proliferation and viable cell numbers, 50,000 YAP C cells were plated in each well of a 6-well plate and transfected as described above. The number of viable cells was determined by counting in a Neubauer counting chamber after 24 to 120 hours of incubation with trypan blue exclusion staining. The results are shown in Figure 2. The proliferation of YAP C cells was inhibited by KRAS1 and depended on the concentration of KRAS1. Only 1 nM of KRAS1 resulted in a statistically significant reduction in the number of viable cells (p=0.001 compared to untreated controls after 120 hours).

Transfection of KRAS2 dsRNA complementary to K-ras wild-type at a concentration of 100 nM resulted in a reduction in the number of viable cells. Non-malignant human dermal fibroblasts transfected with KRAS1 or KRAS2 showed no change in their proliferative behavior.

For in vivo experiments, human pancreatic adenocarcinoma tissue fragments 2-3 mm in diameter were implanted subcutaneously into NMRI mice (Harlan Winkelmann GmbH, Borchen). After the tumor grew to a size of 6-7 mm, KRAS1' or NEO dissolved in physiological saline solution was injected intraperitoneally at a dose of 200 μg per kg body weight daily. Physiological saline solution was injected as a control. Tumor size was measured daily with calipers or a standard template. Figure 3 shows the measured tumor volumes in mm ³ as mean ± standard error of the mean, depending on the time of measurement (days, ip) expressed in days from the start of treatment with intraperitoneal injection. It can be seen from this that dsRNA complementary to K-ras gene can inhibit tumor growth. Tumor growth was inhibited by daily intraperitoneal application of dsRNA complementary to the K-ras gene at a dose of 200 μg/kg, such that after 24 days of treatment, the tumor volume was only 62% of that seen in the control group.

Sequence Listing

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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.

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