WO2012045469A1

WO2012045469A1 - Coated balloon catheter

Info

Publication number: WO2012045469A1
Application number: PCT/EP2011/005012
Authority: WO
Inventors: Manfred GÜLCHER; Georg GÖLLMANN; Hans Peter Wendel; Tobias Walker; Andrea Nolte
Original assignee: Qualimed Innovative Medizinprodukte Gmbh
Priority date: 2010-10-07
Filing date: 2011-10-07
Publication date: 2012-04-12
Also published as: DE102010047588A1; WO2012045469A9

Abstract

The invention relates to a coated balloon catheter for applying pharmaceutically active agents to blood vessel walls, which balloon catheter has a coating with siRNA molecules that are suitable for inhibiting the uncontrolled expression of adhesion molecules, wherein the siRNA molecules are present as polyethyleneimine complex and are embedded in a gelatin matrix.

Description

The invention relates to a coated balloon catheter for the application of pharmaceutically active agents to blood vessel walls.

Arteriosclerosis of the coronary arteries is a common heart disease, especially in developed countries, with serious health consequences, including death. Deposits on the walls of the coronary arteries lead to a narrowing and thus to a limitation of the oxygen supply of the heart muscle. Angina pectoris with resulting chest pain and heart attacks are the result.

In addition to a drug treatment of the deficient circulation invasive treatment methods are indicated. In bypass surgery, sclerotic constricted coronary arteries are replaced by the body's own "fresh" vessels, restoring blood flow, and angioplasty is used to dilate the stenotic coronary vessel, usually by inflating a balloon catheter, causing the vessel lumen is to be expanded - often in combination with the implantation of a stent.

In angioplasty and in particular implantation of stents, but also as a result of surgical interventions, restenoses often form, which in a short time lead to renewed vasoconstriction. To prevent such restenosis, the vascular walls are often acted upon at vulnerable sites with antiproliferative drugs, such as

CONFIRMATION COPY Rapamycin or paclitaxel. These are medicines (immunosuppressants or cytostatics) that were originally developed for the treatment of cancer.

Endothelial cells play an important role in arteriosclerosis and post-treatment restenosis. To prevent, in particular, restenosis, it is therefore necessary to prevent or limit the growth of the subendothelial smooth muscle cells of the vessel wall. Since the vascular system as a whole forms very large surfaces, it is necessary to limit the administration of growth-limiting drugs locally.

In the local application of immunosuppressive agents such as rapamycin and cytostatics such as paclitaxel, the active ingredient is applied in a suitable matrix via a coated balloon or stent to the surface of the vascular area to be treated here. However, such an application presupposes that the active substance remains available locally at least for a certain time and is not washed off by passing blood. This is widely the case with the above-mentioned substances because of their low solubility.

Stenotic changes of arterial vessels are closely related to the damage of endothelial cells of arterial vessels. Injury or inflammation of the vessel walls leads to endothelial activation, which in turn results in increased production of adhesion molecules. The adhesion molecules, in turn, mediate transendothelial migration of leukocytes that affect the integrity of the endothelium. One of these adhesion molecules is E-selectin found on human primary endothelial cells. Selectins are calcium-dependent transmembrane glycoproteins, which play a key role in leukocyte-endothelial interactions in the early phase of arterogenesis. Further examples of adhesion molecules are ICAM-1 (CD54) and VCAM-1 (CD106), see WO 2006/077112 A2. siRNAs (small enterfering RNAs) are short double-stranded RNA molecules, typically containing 21 to 23 nucleotides, that are capable of blocking the translation of a target mRNA. They can methylate specific genes and thereby switch off. From DE 10 2005 003 788 A1 and WO 2006/077112 A2 siRNA molecules are known which are suitable for blocking the expression of adhesion molecules, including the expression of E-selectin. E-selectin, also known as CD62E, is a cell adhesion molecule expressed exclusively from cytokine-activated endothelial cells. It plays an important role in inflammation.

In WO 2006/077112 further oligonucleotides (sense and antisense) under the code numbers SEQ. 1 to SEQ. 44, which are suitable for suppressing the expression of adhesion molecules (ICAM-1, VCAM-1, E-selectin). However, these siRNAs are not readily suitable for local application for the suppression of stenoses.

In view of this, the object of the invention is to provide an application form for siRNA molecules with which these molecules are administered locally in order to inhibit the expression of adhesion molecules on endothelial cells. This object is achieved with a coated balloon catheter of the type mentioned above, which has a coating with siRNA molecules which are suitable for inhibiting the uncontrolled expression of adhesion molecules, the siRNA molecules being present as a polyethyleneimine complex and embedded in a gelatin matrix. The balloon is a conventional dilatation balloon coated according to the invention.

It has been found that the formulation of the invention for the acting in the pharmaceutical sense siRNA molecules among the Application conditions well adhered to the balloon surface can be expanded with this against the vessel wall at the desired location and can be transferred from the balloon surface to the vessel wall so that the siRNA molecules can enter the pending endothelial cells. In parallel, a stent can be placed over the balloon catheter. siRNA molecules are capable of forming complexes with polyethyleneimine (PEI), a synthetic cationic polymer, in which one or more of the oligonucleotides are attached to the polymer chain. These complexes are characterized by their nitrogen / phosphorus ratio (N / P ratio). As a result of the complexation, the siRNA molecules lose nothing of their knock-out effect on the target gene, for example the SELE gene, but can be embedded well into the gelatin matrix via the complex structure and are protected against being flushed off by passing blood. From the gelatin matrix and the complex, the siRNA exerts its effect ("silencing") with high efficiency.

The siRNA molecules are those that are capable of inhibiting the expression of adhesion molecules of human primary endothelial cells. In particular, it is the inhibition of the expression of E-selectin. Such inhibition is achieved in particular with siRNA molecules of the nucleotide sequences siSELE-1 and siSELE-2: siSELE-1: 5 GGU UGA AUG CAC CAC UCA AdTdT-3 '(sense) siSELE-2: 5'-UUG AGU GGU GCA UUC AAC CdTdG-3 '(antisense)

These sequences siSELE-1 and siSELE-2 are also referred to below as siSELE. Sequences derived therefrom having the same properties, which are thus suitable for inhibiting the expression of E-selectin, are likewise encompassed by the invention.

Other siRNA molecules that can be used according to the invention are those mentioned in WO 2006/077112 A2 - see Table 1. On the Revelation of WO 2006/077112 A2 is incorporated by reference, a representation of siRNA oligonucleotides is found in Table 1 below.

For the transfection of the siRNAs to the cells, preferably a synthetic cationic polyethyleneimine is used. This, due to its high cationic charge density, is able to form non-covalent complexes with negatively charged nucleic acids. Under physiological conditions, these complexes are capable of delivering their nucleic acid load to the endothelium where it exhibits the desired effect.

The polyethyleneimine used according to the invention is preferably a branched polyethylenimine having a molecular weight of <50 kD and preferably <30 kD.

The polyethyleneimine may be a low molecular weight polyethyleneimine, such as a molecular weight of 500 to 1,000 D, or one of high molecular weight, especially in the range of 20 to 30 kD. In the case of polyethyleneimine complexes with LMW-PEI (low molecular weight polyethyleneimine), the ratio N / P is preferably less than 14 and in particular 3 to 14. The N / P ratio denotes the ratio of the N atoms in the PEI to the phosphorus atoms of the siRNA.

When the polyethyleneimine complex is a high molecular weight polyethylenimine (HMW) PE, the N / P ratio is preferably in the range of 1 to 8.

The coating of complex applied to the balloon catheter according to the invention contains the siRNA in an amount of 4 to 40 g, preferably 8 to 20 mg in 1 ml of 0.1% gelatin.

In selecting the gelatin, bovine gelatin of (type B) has proved particularly suitable. Gelatine from other species proved to be less well suited.

The invention is explained in more detail by the following examples. PEI-siRNA complex Herstellunq

Complexes of PEI and siRNA were obtained by diluting the siRNAs and PEI - separately - in 150 mM NaCl, 20 mM Hepes, pH 7.4 for 10 min. N / P ratios of 3, 6 and 8 were set based on the assumption that 1 μg siRNA contains 3 mM phosphate and 1 μM 10 mM PEI solution contains 10 mM amine nitrogen. After 10 minutes, the siRNA and PEI solutions were mixed thoroughly and incubated for a further 10 minutes. Thereafter, the mixture was added to 500 μΙ to 0.1% gelatin solution per well of a 2-well microtiter plate and dried for use as a coating, or used for general transfection.

The branched polyethylenimine used was a LMW-PEI from 800 D from Sigma-Aldrich and a HMW-PEI from 25 kD, likewise from Sigma-Aldrich. The gelatin was a bovine type B gel from Fluka.

Cultivation of Endothelial Cells on Coating with Immobilized siRNA After drying the coatings, human endothelial cells (ECs) were seeded in an amount of at least 100,000 cells per well of a microtiter plate with 12 wells. As a medium, EnGS cell culture medium (VascuLife ™) was used with adjuvants, serum and antibiotics. After four hours, the medium was changed to fresh medium. In all experiments, expression of the E-selectin receptor protein was measured by flow cytometry 48 hours after sowing.

Conventional transfection of endothelial cells

One day prior to transfection, ICs were seeded into the wells of a microtiter plate on EnGS cell culture medium (VascuLife ™). After 70% coverage (confluence), the cells were transfected with pg siRNA in the form of the PEI complex at an N / P ratio of 8 in 50 μL of 150 mM NaCl, 20 mM Hepes, pH 7.4. In this case, 50 μΙ complex were padded onto 350 μΙ EnGS base medium, free of additives or medium, and cells were added in a microtiter plate. After two hours, the medium was changed to EnGS cell culture medium.

Influence of serum on siRNA delivery ICs were cultured for 12 hours on EnGS complete cell culture medium (with adjuncts and serum) on coatings. The coatings contained siRNA-PEI complex with an N / P ratio of 8 and 2, 4 or 6 ig siRNA per well. After a culture time of 12 hours, the ICs adhered to the surface of the coating. ... the cells were exchanged for 4 h culture medium against EnGS cell culture medium without serum and additives, then returned to the original culture medium.

Effect of coating components in solution siRNA-PEI complexes with an N / P ratio of 8 and 2, 4 and 6 siSELE per well of a microtiter plate were prepared and added to gelatin. Half of the solution was used as a coating to deliver siRNA to ICs, the other half was mixed with 1 ml of EnGS cell culture medium with adjuncts and serum and added to free ECs in solution. After four hours of culture, the medium was replenished with EnGS cell culture medium supplement and serum. Optimization of siRNA delivery by different coating methods

PEI-siRNA complexes were prepared with N / P ratios of 3, 6 and 8 with 10, 12 and 14 sigELs, 4, 6 and 8 sigELs and 2, 4 and 6 sigELES, respectively. These solutions were each added to 500 μΙ 0.1% gelatin in each well of a microtiter plate. The cells were cultured on the coatings for 48 h and E-selectin expression was determined by flow cytometry.

You're going to love me Forty-eight hours after transfection by coating or general transfection, the ICs were stimulated with 2.5 ng / ml TNF-α (immunotools) to induce E-selectin expression. The cells were stained for 1 h at 4 ° C with a PE-Cy5-labeled E-selectin antibody (Becton Dickenson GmbH) in 0.5% FCS. After washing and detachment of the cells, they were fixed with 2.5% paraformaldehyde in PBS. Flow cytometric analyzes (5,000 cells / per measurement) were made in a FACSscan ™ (Becton Dickenson GmbH) and evaluated with the CellQuestPro software of the same company. The geometric mean values were used for the further analyzes.

Results

The results are explained in more detail in the accompanying figures. Show it:

Figure 1 shows the influence of the on the Herups

Transfection efficiency of the coating;

FIG. 2 shows a comparison of the transfection efficiency of endothelial cells with a coating according to the invention in comparison to the coating components dissolved in the medium;

Figure 3, the release of siRNA

Coatings over time;

Figure 4 shows the extent of inhibition for LNW-PEI siRNA complexes with different N / P ratios in conventional transfection and

FIG. 5 shows the results for HMW-PEI siRNA

Complexes with different N / P

Conditions with conventional transfection. FIG. 1 shows a high transfection efficiency for PEI-siRNA complexes immobilized in gelatin, in the presence as well as in the absence of serum. The reference is the E-selectin expression after application of TNF-a, the value of which was set to 100. Best results were obtained with 6 siRNA (siSELE) in 500 μΙ 0.1% gelatin at an N / P ratio of 8 of the PEI complex. scrRNA is a reference RNA.

Figure 2 compares the transfection efficiency of gelatin-immobilized complex compared to a solution containing the complex components dissolved. The transfection of the endothelial cells with the immobilized siRNA (siSELE) is much more efficient, in fact, the dissolved siRNA shows no significant reduction of E-selectin expression.

FIG. 3 shows a diagram of the release of the complexed siRNA from the gelatin matrix. Most of the siRNA is released from the matrix after 30 minutes and reaches the target cells. The silencing effect lasts longer than 1 week thereafter.

FIG. 4 shows the result of transfection of endothelial cells with LMW-PEI-siRNA (1 [sigma] siRNA). On the X-axis, the different N / P ratios are plotted from 1 to 20, the Y-axis shows the E-selectin receptor training in percent, with the stimulation of cells with TNF-α was set to 100%. High N / P ratios give a very good effectiveness. However, from a ratio of N / P = 14 toxic effects occur, as can be seen from the morphology of the endothelial cells (see upper part of the figure). The epithelium loses its typical plaster structure. FIG. 5 shows the same results of the investigations with HMW-PEI-siRNA complexes with very good results for an N / P ratio of 1 and 3. Toxic effects resulted from N / P = 8. Table 1

siRNA target oligonucleotides SEQ

ID no.

1 ICAM-1 5 '-GCCUCAGCACGUACCUCUA-3' (meaning) 1

5 '-UAGAGGUACGUGCÜGAGGC-5' (antithesis) 2

2 ICA -1 5 '-CCCUUGAÜGAUAUGÜAÜÜU-3' (meaning) 3

5 '-AAAUACAÜAÜCAUCAAGGG-5' (antithesis) 4

3 ICAM-1 5 '-GCCAGCUUAACACAAGAA-3' (meaning) 5

5 '-UUCUUGUGUAUAAGCUGGC-5' (antithesis) 6

4 ICAM-1 5 '-GAUCAAGAAAUACAGACUA-3' (meaning) 7

5 '-UAGUCUGUAUUUCUUGAUC-5' (antithesis) 8

5 ICAM-1 5'-CAAGAAAUACAGACUACAA-3 '(meaning) 9

5 '-UUGUAGUCUGUAUUUCUUG-5' (antithesis) 10

6 ICAM-1 5 '-AGACÜACAACAGGCCCAAA-3' (meaning) 11

5 '-UUUGGGCCÜGUUGUAGUCU-5' (antithesis) 12

7 ICAM-1 5 '- GUCAGAÜACAACAGCAUUU-3' (meaning) 13

5 '-AAAüGCüGÜUGUAUCüGAC-5' (antithesis) 14

8 VCAM-1 5 '-GAUAGAÜAGUCCACUGAAU-3' (meaning) 15

5 '-AUUCAGÜGGACUAUCUAUC-5' (opponent) 16

9 VCAM-1 5 '-GGAUACGGAUAUGAAAUCU-3' (meaning) 17

5 '-AGAUUCAUAUCCGUAUCC-5' (opponent) 18

10 VCAM-1 5 '-GUACGCAAACACUUUAUGU-3' (meaning) 19

5 '-ACAÜAAAGUGUUUGCGUAC-5' (Gegensin) 20

11 VCAM-1 5 '-AAUGCAACUCUCACCUUAA-3' (meaning) 21

5 '-UUAAGGUGAGAGUUGCAUU-5' (antithesis) 22

12 E-selectin 5 '-GACCAUCAAUAAUUACACU-3' (meaning) 23

5 '-AGUGUAAUUÜUGAUGGUC-5' (antithesis) 24

13 E-selectin 5 '-ACGUGUAAAGCUGUGACAU-3' (meaning) 25

5 '-AUGUCACAGCUUUACACGÜ-5' (antithesis) 26

14 E-selectin 5 '-UUAAAGAGAGUGGAGCCÜGGU-3' (meaning) 27

5 '-ACCAGGCÜCCACÜCUCUUÜAA-5' (antithesis) 28 VCAM-1 5 '-GGAGGAUACGGAUAUGAGAAA- 3' (meaning) 29

5 '-UUUCAUAUCCGUAUCCUCC- 3' (antithesis) 30

VCAM-1 5 '-GAGCUAAAUUACACAUUGA- 3' (meaning) 31

5 '-UCAAUGUGUAAUUUAGCUC- 3' (antithesis) 32

VCAM-1 5 '-CAUCUACGCUGACAAUGAA- 3' (meaning) 33

5 '-UUCAUUGUCGCGUAGAUG- 3' (antithesis) 34

VCAM-1 5 '-CUCÜAÜAÜUUAGAÜUGÜUA- 3' (Sinn) 35

5 '-UAACAAUCUAAAUAUAGAG- 3' (antithesis) 36

E-selectin 5 '-GGUGAAACACCACUCAA- 3' (meaning) siSELE-1

5 '-UUGAGUGGUGCAUUCAACC- 3' (antithesis) -2

E-selectin 5 '-UGGUAGAAUUGGAGAGUAA- ■ 3' (meaning) 39

5 '-UUAC-BACKUP-BACKACCA- -3' (antithesis) 40

E-selectin 5 '-CAGUGUGGUUUGUGUUGA- -3' (Meaning) 41

5 '-ÜCAAACACAAACCACACÜG- ■ 3' (antithesis) 42

E-selectin 5 '-CGGAAGCUAUGACUUAUGA-' 3 '(meaning) 43

5 '-REVERSEAGUCAÜAGCUUCCG- • 3' (antithesis) 44

Negative5 '-ÜUCUCCGAACGÜGÜCACGU- -3' (Sinn) 45 control 5 '-ÄCGÜGACACGÜÜCGGAGAA- ■ 5' (Gegensinn) 46

Claims

claims

A coated balloon catheter for applying pharmaceutically active agents to blood vessel walls characterized by coating the balloon with siRNA molecules capable of inhibiting the uncontrolled expression of adhesion molecules, the siRNA molecules being in the form of a polyethyleneimine complex and embedded in a gelatin matrix ,

2. Balloon catheter according to claim 1, characterized in that the siRNA molecules are suitable to inhibit the expression of adhesion molecules of human primary endothelial cells.

3. Balloon catheter according to claim 1 or 2, characterized in that the siRNA molecules inhibit the expression of ICAM-1, VCAM-1 and / or E-selectin.

4. Balloon catheter according to one of the preceding claims, characterized in that the siRNA molecules have one of the nucleotide sequences of Tab. 1 or a sequence derived therefrom having the same properties.

5. Balloon catheter according to one of the preceding claims, characterized in that the polyethyleneimine is a branched polyethylenimine having a molecular weight of <50 kD, preferably <30 kD.

6. Balloon catheter according to claim 5, characterized in that the polyethyleneimine has an LMW-PEI having a molecular weight of 500 to 1000 D.

7. Balloon catheter according to claim 6, characterized in that the Polyethyleniminkomplex has an N / P ratio of <14, preferably from 3 to 14.

8. Balloon catheter according to one of claims 1 to 5, characterized in that the polyethyleneimine complex is a HMW-PEI having a molecular weight of 20 to 30 kD. 9. Balloon catheter according to claim 8, characterized in that the Polyethyleniminkomplex has an N / P ratio of 1 to 8.

9. Balloon catheter according to one of the preceding claims, characterized in that the coating contains 4 to 40 pg, in particular 8 to 20 pg siRNA per 1 ml of 0.1% gelatin.

10. Balloon catheter according to one of the preceding claims, characterized in that the gelatin is a bovine type B gelatin.