Balloon catheter with a sirolimus coated catheter balloon for controlled release of sirolimus

Abstract

The present invention is directed to a catheter balloon of a balloon catheter comprising a coating consisting of or containing at least one fatty acid, oil or fat and sirolimus as well as to the use of these systems and compositions. Optionally a stent is crimped on such a coated catheter balloon. Further, methods for coating the inventive catheter balloons are described.

Claims

1. A catheter balloon with a coating consisting of sirolimus, an omega-3fatty acid and shellac.

2. The catheter balloon according to claim 1, wherein the omega-3 fatty acid is chosen from the group consisting of: eicosapentaenoic acid, eicosatrienoic acid, eicosatetraenoic acid, docosahexaenoic acid, hexadecatrienoic acid, stearidonic acid, heneicosapentaenoic acid, docosapentaenoic acid, tetracosapentaenoic acid, tetracosahexaenoic acid and -linolenic acid as well as mixtures of the aforementioned fatty acids.

3. A balloon catheter comprising a catheter balloon according to claim 1.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1: Intramural Paclitaxel and Sirolimus median concentration [g/g]

(2) FIG. 2: Residuals of Paclitaxel and Sirolimus on catheter balloons after balloon angioplasty in pigs (example 16)shown is the average residual in percentage of total loading dose

(3) FIG. 3: Mean Tissue sirolimus concentration [ng/mg] 1 hour after dilatation of the balloon of example 17.

EXAMPLES

Example 1

Coating with Sirolimus and -Linolenic Acid

(4) Sirolimus is solved in DMSO containing ca. 10 vol. % of water. -Linolenic acid and shellac are added to this solution and the catheter balloon is coated several times with this solution by using the spraying method (e.g. EVOLUTION spraying pistol from Harder & Steenbeck) and dried after the coating.

Example 2

Coating of the Folds of a Catheter Balloon Only

(5) A mixture of -linolenic acid, polyethoxylated castor oil (2:1) and sirolimus in ethanol and water is prepared, filled into a pipette and squirted by means of the pipette under the folds of a multifold balloon. After drying a powdery coating of the fold interspaces results, which is easily detached on dilating the balloon.

Example 3

Coating of a Catheter Balloon with Shellac and -Linolenic Acid (1:3)

(6) A mixture of -linolenic acid and shellac (3:1) in ethanol and water is prepared, filled into a pipette and squirted by means of the pipette on the complete surface of a catheter balloon. Afterwards the catheter balloon was dried for 24 h at room temperature.

Example 4

Coating of a Catheter Balloon Sirolimus and Stearidonic Acid

(7) A common balloon catheter with an expandable catheter balloon, which may be used for vessel dilatation, is removed from fat in the ultrasonic bath for 15 minutes with acetone and ethanol and dried at 100 C. in the drying oven until acetone and ethanol are evaporated. Subsequently the catheter balloon is washed with demineralized for 12 hours. 10 mg of Sirolimus are solved in 1 mL of ethanolic solution of stearidonic acid (Cayman Chemical). The mixture is sprayed with an airbrush spraying pistol from a distance of 5.8 cm on a rotating 18 mm LVM balloon catheter. Afterwards the coated balloon was dried for 24 h at 30 C. Optionally the coated catheter balloon may be folded and an uncoated or active agent-coated stent may be crimped on the folded catheter balloon.

Example 5

Adding of Sirolimus to a Coated Balloon in the Dipping Method Using Sterile Conditions

(8) The catheter balloon coated according to example 3 was dipped into a solution of 300 g of sirolimus in 1 ml of ethanol and allowed to swell. After accomplishing the swelling process of the coating the catheter balloon was pulled out of the solution, air dried for 120 minutes at room temperature and folded. Optionally an uncoated or active agent-coated stent may be crimped on the folded catheter balloon.

Example 6

Biocompatible Coating of a Catheter Balloon with Linseed Oil, Sirolimus and Paclitaxel

(9) A common balloon catheter with an expandable catheter balloon, which may be used for vessel dilatation, is removed from fat in the ultrasonic bath for 15 minutes with acetone and ethanol and dried at 100 C. in the drying oven until acetone and ethanol are evaporated. Subsequently the balloon catheter was washed with demineralized water for 14 hours. Linseed oil, sirolimus and paclitaxel (80:10:10 percent by weight) are dissolved in the mixture ratio of 1:1 in chloroform after the resulting volume has been measured. Subsequently the mixture is sprayed on the balloon surface of the continuously rotating balloon catheter. After evaporation of the chloroform in the soft air stream the balloon catheter is dried at 80 C. Optionally the coated catheter balloon may be folded and an uncoated or active agent-coated stent may be crimped on the folded catheter balloon.

Example 7

Biocompatible Coating of a Catheter Balloon with an Ethanolic Linseed Oil Spraying Solution with 0.25 Percent by Weight of Linseed Oil

(10) A common balloon catheter with an expandable catheter balloon, which may be used for vessel dilatation, is removed from fat in the ultrasonic bath for 15 minutes with acetone and ethanol and dried at 100 C. in the drying oven. Subsequently the balloon catheter was washed with demineralized water for 14 hours. A spraying solution of linseed oil, sirolimus and ethanol is prepared and continuously sprayed with a spraying pistol on the balloon surface of the balloon catheter rotating around its axis. The balloon catheter with the coated balloon is dried for 13 hours at 70 C. The average coating mass is 0.20 mg0.04 mg. Optionally the coated catheter balloon may be folded and an uncoated or active agent-coated stent may be crimped on the folded catheter balloon.

Example 8

Biocompatible Coating of a Catheter Balloon with an Ethanol Spraying Solution of Gamma-Linolenic Acid, Polyethoxylated Stearic Acid and Sirolimus

(11) After cleaning of the catheter balloons as already described in the example 6 an ethanol spraying solution is prepared which contains 0.25% gamma-linolenic acid and 0.1% polyethoxylated stearic acid. Subsequently, sirolimus is solved in this ethanolic solution. The ethanolic solution is continuously sprayed with a spraying pistol on the balloon surface of a rotating balloon catheter, which is suitable for vessel dilatation. Then the balloon catheter is dried for 15 hours at 70 C. The average coating mass is 0.3 mg0.06 mg. Optionally the coated catheter balloon may be folded and an uncoated or active agent-coated stent may be crimped on the folded catheter balloon.

Example 9

Coating of a Catheter Balloon with Sirolimus, -Linolenic Acid and Shellac in a Two Layer System with Addition of a Top Coat

(12) The balloon of a balloon catheter is cleaned as described in the example 6. After cleaning a first layer of 0.25% by weight of sirolimus, -linolenic acid and shellac dissolved in DMSO is continuously sprayed on the balloon surface of a rotating balloon catheter. This layer is dried for 4.5 hours at room temperature. Subsequently the second layer of an ethanol solution with 0.1% polyvinyl alcohol-polyethylene glycol graft copolymer is sprayed on this first layer. The balloon catheter with the coated balloon is dried over 24 hours at 50 C. The average coating mass is determined to be 0.25 mg0.02 mg. Optionally the coated catheter balloon may be folded and an uncoated or active agent-coated stent may be crimped on the folded catheter balloon.

Example 10

Coating of a Catheter Balloon with Sirolimus, -Linolenic Acid and Shellac in a Two Layer System with Addition of a Base Coat

(13) The balloon of a balloon catheter is cleaned as described in the example 6. After cleaning of the balloon a first layer of 0.3% by weight of shellac dissolved in ethanol is sprayed on the balloon surface of a balloon catheter. This layer is dried at room temperature over 12.5 hours. Subsequently, the second layer of a DMSO solution with 0.25% by weight of sirolimus, -linolenic acid and shellac is sprayed on the base coat of shellac. After drying over 20 hours at 30 C. the coating mass is determined to be 0.42 mg0.07 mg. Optionally the coated catheter balloon may be folded and an uncoated or active agent-coated stent may be crimped on the folded catheter balloon.

Example 11

Coating of a Catheter Balloon with Linseed Oil and -Linolenic Acid

(14) A common balloon catheter with an expandable catheter balloon, which may be used for vessel dilatation, is removed from fat in the ultrasonic bath for 15 minutes with acetone and ethanol and dried at 100 C. in the drying oven until acetone and ethanol are evaporated. Then a mixture of 0.20% per weight linseed oil and 0.5% per weight -linolenic acid solved in ethanol is prepared. This mixture is continuously sprayed on the balloon surface of a rotating balloon catheter. A drying step takes place over 14 hours at 70 C. The average coating mass is 0.3 mg0.04 mg. Optionally the coated catheter balloon may be folded and an uncoated or active agent-coated stent may be crimped on the folded catheter balloon.

Example 12

Study of Restenosis Inhibition by Sirolimus after Angioplasty and Stent Implantation in the Coronary Arteries of Pigs

(15) The balloon surfaces of balloon catheters suitable for vessel dilatation has been coated as described in table 9. After drying a common, uncoated metal stent was crimped on each balloon catheter. The balloon catheter together with the stents were sterilized packed into protective covers and stored until usage at room temperature.

(16) For stimulation of restenosis caused by tissue hyperplasia coronary vessels (LAD and LCx) of 50 pigs were stretched using a balloon catheter. The animals used were female and castrated male pigs of Yorkshire breed. The weight of the animals at the beginning of the experiment ranged between 24 to 35 kg; the age of the animal was about 12 to 15 weeks. The group size was five animals each.

(17) The vessel dilatation of LAD and LCx using balloon dilatation was performed for 30 seconds in the ratio balloon/artery in the range of 1.2 to 1 and 1.3 to 1 and was repeated once. Thereafter, the crimped stents were implanted into the vessel wall so that a stent was implanted in LAD and LCx of each animal. For the balloon dilatation balloons coated according to the invention were used. Table 9 shows an overview of the used coatings and coating methods.

(18) TABLE-US-00010 TABLE 9 Overview of coatings and stent implantation Group (n = 5) Balloon surface coated with Stent implantation 1 No coating 1 LAD, 1 LC.sub.x 2 Linseed oil + PVP 1 LAD, 1 LC.sub.x 3 -linoleic acid + PVP 1 LAD, 1 LC.sub.x 4 Linseed oil + sirolimus 1 LAD, 1 LC.sub.x 5 Linseed oil + PVP + sirolimus 1 LAD, 1 LC.sub.x 6 Linseed oil + PVP + sirolimus 1 LAD, 1 LC.sub.x (PVP as a top coat) 7 Linseed oil + -linoleic acid 1 LAD, 1 LC.sub.x 8 -linoleic acid + sirolimus 1 LAD, 1 LC.sub.x 9 -linoleic acid + sirolimus + PVP 1 LAD, 1 LC.sub.x (PVP as a top coat)

(19) After 28 days angiography was carried out at the LAD and LCx of the animals. Stenosis grad gives the percent reduction of the lumen diameter in the area of the stent to the lumen diameter immediately after implantation of the stent. Values are expressed as mean valuesstandard deviation. Differences compared to group 1 which was treated with balloon catheters with uncoated balloons were considered significant at a value of P<0.05. The results are shown in Table 10.

(20) TABLE-US-00011 TABLE 10 Results of angiography after 28 days Degree of stenosis % [mean value standard deviation] Group (P-value in comparison to (n = 5) Surface coating group 1) 1 No coating 44.6 17.1 2 Linseed oil + PVP 38.8 19.6 (P = 0.490) 3 -linoleic acid + PVP 37.2 18.0 (P = 0.358) 4 Linseed oil + sirolimus 23.3 19.4 (P = 0.018) 5 Linseed oil + PVP + sirolimus 17.2 19.3 (P = 0.004) 6 Linseed oil + PVP + sirolimus 13.7 18.1 (P = 0.001) (PVP as a top coat) 7 Linseed oil + -linoleic acid 29.9 24.3 (P = 0.135) 8 -linoleic acid + sirolimus 10.7 15.1 (P = 0.011) 9 -linoleic acid + sirolimus + PVP 9.8 13.5 (P = 0.006) (PVP as a top coat)

Example 13

Determination of the Degree of Inflammation in the Treated Tissue Area after 28 Days

(21) After angiography, the animals of Example 12 were euthanized and coronary tissue samples were taken for histology. The degree of inflammation was evaluated using the following classification: 0 media or intima shows no inflammation o rare interspersed with small amounts of inflammatory cells 1 weak infiltration or moderate inflammatory lesions within less of 25% of the vessel area in the media or intima 2 moderate infiltration or notable inflammatory lesions between 25% to 50% of the vessel area in the media or intima 3 strong infiltration or notable inflammatory lesions within more than 50% of the vessel area in the media or intima 4 granulomatous inflammatory reaction in any layer of the artery

(22) The results of determination the degree of inflammation after 28 days are shown by table 11. Values represent mean valuesstandard deviation. Differences to group 1 which was treated with balloon catheters with uncoated balloons were considered significant at a value of P<0.05. All investigated coatings resulted in no significant increase in levels of inflammation after 28 days.

(23) TABLE-US-00012 TABLE 11 Degree of inflammation after 28 days Degree of inflammation [mean value standard deviation] Group (P-value in comparison to (n = 5) Surface coating group 1) 1 No coating 0.40 0.80 2 Linseed oil + PVP 0.81 0.79 (P = 0.2369) 3 -linoleic acid + PVP 0.80 0.63 (P = 0.2301) 4 Linseed oil + sirolimus 0.70 1.06 (P = 0.4842) 5 Linseed oil + PVP + 0.70 0.82 (P = 0.4185) sirolimus 6 Linseed oil I + PVP + 0.90 1.10 (P = 0.2602) sirolimus (PVP as a top coat) 7 Linseed oil + -linoleic acid 0.60 0.94 (P = 0.6146) 8 -linoleic acid + sirolimus 0.95 0.80 (P = 0.2301) 9 -linoleic acid + sirolimus + 0.8 1.05 (P = 0.006) PVP; (PVP as a top coat)

Example 14

Coating of a Folded Balloon with Oleic Acid and Sirolimus Using Pipetting Method

(24) A common balloon catheter with an expandable, folded balloon, which may be used for vessel dilatation, is removed from fat in the ultrasonic bath for 15 minutes with acetone and ethanol and dried at 100 C. in the drying oven until acetone and ethanol are evaporated. Subsequently, the folded balloon is washed with demineralized water over 12 hours. A mixture of 85% per weight oleic acid and 15% per weight sirolimus is produced and the same volume ethanol is added to the resulting mixture. The folded balloon is tethered in a horizontal position on the rotatable axis so that the fold to be filled is always lying upside. Thus step by step each fold is filled with the coating solution from the beginning to the end of the fold by means of a teflon cannula as enlargement of a needle syringe. Subsequently the coated balloon catheter is dried over night by room temperature until the solvent is evaporated completely. Then a coated or uncoated stent may be crimped on, if desired.

Example 15

Coating of a Folded Balloon with Oleic Acid/Shellac and Sirolimus Using Pipetting Method

(25) The folded balloon of a balloon catheter is cleaned and coated as described in example 13. But the coating solution is produced using 40% per weight oleic acid, 40% per weight shellac and 20% per weight sirolimus. The same volume ethanol is added to the resulting mixture.

Example 16

Pharmacokinetic Evaluation of Sirolimus Eluting, Hydrophilically Coated Balloons in an Overstretch Model of Porcine Coronary Arteries

(26) 1Material and Methods

(27) Eight Polish Domestic pigs of 35-42 kg body weight were included in the study in which 24 sirolimus and paclitaxel eluting balloons (, cf. above) were deployed. Procedures were carried out in the Center for Cardiovascular Research of American Heart of Poland. The appropriate approval of regional Bioethical Committee was obtained. The three coronary arteries (LAD, LCx, RCA) of each animal were randomly assigned in 1:1:1 ratio to either study group or reference group. All animals received dual antiplatelet therapy consisting of oral acetylsalicylic acid (325 mg) and clopidogrel (300 mg initial dose and 75 mg subsequently) starting three days prior to intervention and continuing until sacrifice. After anesthesia induction with propofol the animals were intubated and supported with mechanical ventilation. A propofol continuous infusion was started to maintain a surgical plane of anesthesia. Subsequently, an arterial sheath was introduced to the left or right femoral artery utilizing percutaneous Seldinger technique. An initial bolus of heparin (400 U/kg) was administered and activated clotting time (ACT) was measured every 30 minutes to maintain ACT time of at least 300 seconds. Coronary angiograms were performed after administration of intracoronary nitroglycerin (200 g). The selection of the target site was made based on visual assessment of anatomy and quantitative coronary angiography (QCA) analysis. These sites were chosen for the avoidance of side branches and segments with tapering greater than 10% to ensure uniform interaction of the stent coating with the arterial wall. The balloon was then inflated at a steady rate to a pressure sufficient to achieve a balloon to artery ratio of 1.2:1 (acceptable range of 1.15:1 to 1.25:1). At pre-determined time points the animals were euthanized. The hearts were harvested as quickly as possible after euthanasia, using precautions to avoid damage to the study vessels. The hearts were examined for abnormal findings and were labeled with the animal identification number, protocol number and date of collection. The hearts were flushed with normal saline until cleared of blood and then pressure-perfusion fixed at 80-100 mmHg with 10% neutral buffered formalin (NBF). Samples of abnormal tissues were collected and undergo immersion fixation with 10% NBF. All study vessel segment were labeled with the animal identification number, protocol number, tissue types and date of collection. All tissues were placed in containers and frozen in dry ice in 68 C. and sent to the HPLC test site. The heart for each animal was placed in its own separate container.

(28) Sirolimus and Paclitaxel Eluting Balloon Catheter Characteristics:

(29) Three studied catheters with the following coatings were evaluated: 1. study group 1: 3.0 g/mm.sup.2 Sirolimus+Shellolic Acid++0.5 g/mm.sup.2 -linolenic acid coated balloon 2. study group 2: 3.0 g/mm.sup.2 Sirolimus+Shellolic Acid coated balloon 3. reference group: =3.0 g/mm.sup.2 Paclitaxel+3.0 g/mm.sup.2 Shellac coated balloon All used balloons were 3.0 or 3.5 mm in diameter and 20 mm in length.
Qualitative Coronary Angiography

(30) The Quantitative Coronary Angiography (QCA) analysis was performed by blinded operator with the off line QAngioX 7.2, MEDIS Software and angiograms were recorded in DICOM format. Two contralateral projections were chosen for balloon deployment site assessment.

(31) HPLC Analysis (Appendix 1)

(32) The paclitaxel or sirolimus concentration of plasma, LAD, LCx and RCA was measured by high-performance liquid chromatography (AnaKat Institut for Biotechnologie GmbH, Berlin, Germany, analysis blinded to sample origin). Briefly, after thawing, the tissues were weighed at ambient temperature and, depending on the weights; different volumes of ethanol were added to the samples (sufficient ethanol to cover the tissue completely). The samples were then treated with ultrasound for 40 minutes. About 200 ml samples were centrifuged. A calibration line was produced in the range between 50 and 5000 ng/ml. The samples for the calibration line were prepared by dilution of a stock solution with a concentration of 1000 mg/ml. Aliquots of all samples (samples from tissue and calibration line) were transferred into autosampler vials and the same volume of 0.1% formic acid was added. The flow rate of the high performance liquid chromatography system was 0.2 ml/min through a column of ODS Hypersil (ThermoElectron Corporation, Thermo Scientific, Waltham, Mass., USA), particle size 5 m, pore size 120 A.sup.. The isocratic mobile phase consisted of 70% methanol containing formic acid (0.1%). Paclitaxel was detected by mass spectrometry in multiple reaction-monitoring mode with a transition of paclitaxel from 854 to 105 AMU. The tissue paclitaxel/sirolimus concentration was expressed in g/g.

(33) Follow-Up

(34) The animals were scheduled for 1 hour, 1, 3 and 7 days (2 pigs per each time period) according to table 12.

(35) TABLE-US-00013 TABLE 12 scheme of balloon angioplasty Animal no. Follow up (days) Group Artery 1 7 Study group_1 lad 1 7 Study group_2 lcx 1 7 Reference group rca 2 7 Reference group lcx 2 7 Study group_2 lad 2 7 Study group_1 rca 3 3 Study group_2 rca 3 3 Study group_1 lcx 3 3 Reference group lad 4 3 Study group_1 lcx 4 3 Reference group lad 4 3 Study group_2 rca 5 1 Study group_2 rca 5 1 Study group_1 lad 5 1 Reference group lcx 6 1 Study group_2 lcx 6 1 Study group_1 lad 6 1 Reference group rca 7 0 Study group_1 rca 7 0 Study group_2 lcx 7 0 Reference group lad 8 0 Reference group lcx 8 0 Study group_2 lad 8 0 Study group_1 rca
Statistical Analysis:

(36) Results are expressed as meanstandard deviation (SD). Normal distribution of variables was verified by Kolmogorov-Smimov test. The variance uniformity was verified with the use of Levene test. Angiographic and HPLC analysis data were analyzed using ANOVA tests. In case of skewed distribution or non-uniformity of variance a nonparametric Kruskal-Wallis and U Mann-Whitney tests were used. The p-value<0.05 was considered statistically significant. Statistical analysis was performed utilizing Statistica 7.0 StatSoft Software.

(37) Results

(38) Pre-Operative Procedures

(39) After an overnight fast, the animals were pre-anesthetized with a mixture based on body weight. These active agents include: Atropine (1 mg/20 kg sc.), Ketamine (1 ml/10 kg im) and Xylazine (1 ml/10 kg im). The injection was given intramuscularly (im) in either the neck or rear muscle quadrant by a qualified animal technologist. The animal was transferred to the preparation room, where an intravenous line was placed in the auricular marginal vein, and intravenous fluids (lactated ringers or 0.9% saline) were administered throughout the procedure. Anti-arrhythmics were added to these IV fluids (Lidocaine 200 mg/liter, Metoprolol 5 mg/liter). After the animal reached an adequate anesthetic plane (with a propofol bolus) it was intubated with an appropriate size endotracheal tube, which was tied into place and the cuff inflated to prevent leakage. The animal was then transferred to the catheter lab, placed on the table and attached to the anesthesia and ventilator unit.

(40) Procedures

(41) In total 24 balloons were deployed, eight Study group 1 (five 3.5 mm and three 3.0 mm), eight Study group 2 (three 3.5 and five 3.0 mm) and 8 Reference group as shown in Table 12. Each of them was inspected before delivery. No signs of structure abnormality were noticed. The coating was not visible. Balloons were easily introduced into the selected arterial segment through femoral artery access and successfully deployed in the desired segment after live QCA guidance to ensure balloon/artery ratio 1.2:1. All tested balloons were inflated for 60 s. Due to overstretch in one case a dissection, limiting blood flow occurred. This required a stent implantation proximally to the tested site.

(42) Baseline Vessel and Balloon Deployment Characteristics:

(43) There were no differences in the baseline QCA results such as vessels baseline reference diameter, minimal lumen diameter, balloon diameter and stent to artery ratio in the whole group as well as within each time period between the studied groups (Table 13). The average overstretch was 110-120% and was reproducible among groups. All tested balloons stayed in circulation for 3 minutes20 seconds

(44) TABLE-US-00014 TABLE 13 Baseline QCA vessel characteristics [mm] RD MLD Balloon S-2-A All Average SD Average SD Average SD Average SD Total n = 24 STUDY GROUP 1 2.72 0.36 2.93 0.39 3.07 0.25 1.14 0.13 STUDY GROUP 2 2.71 0.35 2.92 0.33 3.00 0.27 1.10 0.09 REFERENCE 2.60 0.40 2.78 0.38 2.96 0.28 1.15 0.08 GROUP P ANOVA ns ns ns ns 1 hour (0) n = 6 STUDY GROUP 1 2.88 0.45 3.01 0.42 3.11 0.21 1.10 0.24 STUDY GROUP 2 2.44 0.41 2.64 0.51 2.88 0.20 1.19 0.11 REFERENCE 2.81 0.53 2.97 0.48 3.11 0.35 1.12 0.09 GROUP P ANOVA ns ns ns Ns 1 day n = 6 STUDY GROUP 1 2.50 0.15 2.69 0.25 3.05 0.24 1.23 0.02 STUDY GROUP 2 3.06 0.35 3.23 0.28 3.28 0.27 1.08 0.04 REFERENCE 2.27 0.21 2.43 0.24 2.81 0.25 1.24 0.00 GROUP P ANOVA ns ns ns ns 3 days n = 6 STUDY GROUP 1 2.64 0.71 2.85 0.74 2.99 0.43 1.15 0.14 STUDY GROUP 2 2.54 0.18 2.95 0.05 3.00 0.00 1.12 0.00 REFERENCE 2.72 0.31 2.89 0.20 3.03 0.38 1.11 0.01 GROUP P ANOVA ns ns ns Ns 7 days n = 6 STUDY GROUP 1 2.86 0.02 3.19 0.08 3.15 0.36 1.10 0.11 STUDY GROUP 2 2.79 0.28 2.85 0.27 2.84 0.30 1.02 0.00 REFERENCE 2.61 0.59 2.83 0.58 2.88 0.34 1.12 0.13 GROUP P ANOVA ns ns ns ns
Paclitaxel Concentration Analysis and Follow Up.

(45) Within the entire follow-up period, neither death nor major adverse events cardiac events were noted. All animals remained in good general condition until euthanasia. Delivered paclitaxel concentrations by Reference group (reference balloon) were within the range of 1-12 g/g, with steady decrease over 7 days (FIG. 1).

(46) The balloons of both study groups delivered sirolimus in the range of 1-15 g/g. At one hour follow up each balloon of a study group delivered sirolimus in the amount of 10-20 g/g. At one day follow up in study group 1 sirolimus concentration found in the vessel was beyond 140 g/g. On the other hand the second balloon set for the same follow up did not deliver sirolimusto the vessel wall. In the final observation sirolimus concentration was in the range of 1-1.5 g/g in both studied groups (FIG. 1). None of the recorded differences were statistically significant.

(47) The active agent residuals were significantly higher in the paclitaxel eluting balloons.

(48) TABLE-US-00015 TABLE 14 Vessel intramural Paclitaxel and Sirolimus Concentration in g/g (median and IQR) ug/g Study group 1 N = 2 Study group 2 N = 2 Reference group N = 2 ANOVA P 1 hour 12.8 [9.2:16.3] 14.4 [12:16.7] 10.1 [5.4:14.7] Ns 1 day 63.1 [0:126.2] 8.8 [2.6:15.1] 6.65 [0-13.3] Ns 3 days 0.65 [0:1.31] 0.68 [0.15-1.22] 1.5 [0.1:2.92] Ns 7 days 1.27 [1.25:1.29] 0.75 [0-1.5] 1.65 [0-3.3] Ns

(49) TABLE-US-00016 TABLE 15 Vessel intramural Paclitaxel and Sirolimus Concentration in mol/l (median and IQR) mol/l Study group 1 N = 2 Study group 2 N = 2 Reference group N = 2 ANOVA P 1 hour 15 [10.8:19.1] 16.8 [14.1:19.6] 11.8 [6.3:11.8] Ns 1 day 73.8 [0:147.7] 10.3 [3:17.6] 7.8 [0:15.6] Ns 3 days 0.7 [0:1.5] 0.8 [0.17:1.4] 1.8 [0.1:3.4] Ns 7 days 1.5 [1.5:1.5] 0.9 [0:1.8] 1.9 [0:3.9] Ns

(50) TABLE-US-00017 TABLE 16 Detailed concentration of sirolimus and paclitaxel concentration concentration no. vessel FU Active agent [mol/l] [g/g] 1 RCA 7 Reference group 3.87 3.30 1 LAD 7 Study group 1 1.47 1.26 1 LCX 7 Study group 2 0.00 0.00 2 RCA 7 Study group 1 1.52 1.30 2 LAD 7 Study group 2 1.76 1.50 2 LCX 7 Reference group 0.00 0.00 3 RCA 3 Study group 2 0.17 0.15 3 LAD 3 Reference group 3.42 2.92 3 LCX 3 Study group 1 1.54 1.32 4 RCA 3 Study group 2 1.43 1.22 4 LAD 3 Reference group 0.11 0.10 4 LCX 3 Study group 1 0.00 0.00 5 RCA 1 Study group 2 17.64 15.07 5 LAD 1 Study group 1 0.00 0.00 5 LCX 1 Reference group 0.00 0.00 6 RCA 1 Reference group 15.58 13.30 6 LAD 1 Study group 1 147.77 126.18 6 LCX 1 Study group 2 3.00 2.56 7 RCA 0 Study group 1 10.80 9.22 7 LAD 0 Reference group 17.27 14.75 7 LCX 0 Study group 2 14.05 12.00 8 RCA 0 Study group 1 19.11 16.32 8 LAD 0 Study group 2 19.59 16.72 8 LCX 0 Reference group 6.34 5.42

(51) TABLE-US-00018 TABLE 17 Paclitaxel and Sirolimus residuals on balloons (ug/mm.sup.2) Different (P < 0.05) Factor n Mean from factor nr (1) STUDY GROUP 1 8 99.6 (3) (2) STUDY GROUP 2 8 119.2 (3) (3) REFERENCE GROUP 8 198.8 (1)(2)

CONCLUSION

(52) All tested balloons were easily introduced and deployed at study sites. No delivery or withdrawal problems occurred. The balloon diameters at nominal inflation reached their designed diameter. No adverse events were noted neither after procedures nor at follow up. On autopsy no macroscopic signs of myocardial infarction or inflammation within studied site were noted. It must be noted that due to very short term of observation and design of the study. the safety of studied balloon catheters was not an endpoint.

(53) The baseline studied vessel characteristics between groups were similar with regard to reference diameter and minimal lumen diameter. Most importantly the stent to artery ratio of 1.1-1.2:1 resulted in similar overstretch between the studied groups. On the other hand due to very low number of tested balloons per group in each time point (n=2) a reproducible overstretch numerically was hard to achieve. All inflations were performed for 60 s and all balloon remained within the same period of time in circulation. Basing on previous studies this overstretch and inflation time should definitely provide proper and reproducible conditions for active agent delivery (1,2).

(54) The paclitaxel concentrations delivered by the reference group were within 1-10 g/g. This result is not comparable to currently clinically available paclitaxel eluting balloons which achieved higher tissue paclitaxel concentrations (2,3). Both tested sirolimus balloons delivered sirolimus to the vessel wall within the range of 1.5-20 g/g. In all vessels sirolimus was found in 10-20 g/g after 1 hour, therefore proving very good deliverability of sirolimus into the wall. In one tested study group 1 balloon the vessel concentration of sirolimus achieved an extraordinary result of 142 g/g at 24 hour. Although the overstretch was high in this case (1.24:1) the second study group 1 balloon, deployed with the same inflation pressure and overstretch, did not deliver sirolimus to the vessel. Despite high numerical differences between study group 1 and 2 (63 vs. 8 g/g) in this time point the result did not reach any statistical significance. At 7 days follow up in the study group 1 the concentrations were within 1.27 g/g with a very low interquartile range and therefore are very reproducible.

(55) This study is the first one that proved sirolimus deliverablity to the vessel wall (1) and therefore very promising. In a series of very few and rarely published reports sirolimus as a not liphophilic substance delivered from the balloon did not reach significant concentrations. Because the therapeutic or cytotoxic sirolimus concentrations after sirolimus eluting balloon angioplasty needed for neointima inhibition are unknown a long term tissue effects study is mandatory to prove its safety and efficacy in the porcine model of restenosis.

References for Example 16

(56) 1. Gray W A. Granada J F. Drug-coated balloons for the prevention of vascular restenosis. Circulation; 121:2672-80. 2. Posa A. Hemetsberger R. Petnehazy O. et al. Attainment of local drug delivery with padcitaxel-eluting balloon in porcine coronary arteries. Coron Artery Dis 2008; 19:243-7. 3. Scheller B. Speck U. Schmitt A. Bohm M. Nickenig G. Addition of paclitaxel to contrast media prevents restenosis after coronary stent implantation. J Am Coll Cardiol 2003; 42:1415-20.

Example 17

Proof-of-Principle Study for the Active Agent Transfer of a Sirolimus-Eluting Balloon in a Healthy Rabbit Model

(57) A sirolimus eluting balloon (3.020 mm) coated with a blend of sirolimus (3 g/mm.sup.2), shellac (3 g/mm.sup.2) and -linolenic acid (1.5 g/mm.sup.2) (in this example called DEB) was evaluated in regard to an effective sirolimus transfer into the arterial tissue during inflation of the balloon.

(58) This study was carried out at the Deutsches Herzzentrum Mnchenclinic at the Technical University Munich. The appropriate approval of regional Bioethical Committee was obtained. HPLC-MS-based analysis for sirolimus content in tissue was conducted at ic42 Laboratory, University of Colorado, USA.

(59) Study Design:

(60) A total of 4 sirolimus eluting balloons were deployed in 2 healthy New Zealand White rabbits. For this purpose animals were anaesthetized with propofol and intra surgery analgesia was secured by repetitive boli of fentanyl. Animals were intubated, mechanically ventilated and at all times controlled for vital signs (pulse-oximetry and capnography). Anticoagulation was achieved by administration of 500 IU heparin and 40 mg aspirin i.v. Arterial access was conducted by cut down of the common carotid artery. A swan ganz catheter was advanced over the aortic arch under fluoroscopic guidance just before the bifurcation of the common iliac arteries of the abdominal aorta and an initial angiogram was performed. A guide wire was then placed in the external iliac artery. Wire guided balloon injury (POBA) with single balloon inflation [3.010 mm size balloon (Elect, Biotronik SE & Co. KG) at nominal pressure (7 atm) held for 30 seconds] within the middle portion of the external iliac artery was performed to induce arterial injury and facilitate sirolimus uptake into the vascular wall of healthy arteries. Afterwards the sirolimus eluting balloons were deployed covering the whole length of the induced lesion. The sirolimus eluting balloons were inflated at nominal pressure (6 atm) for 60 seconds. Five minutes after the procedure, a final angiogram was conducted and the animals were kept under anaesthesia until study termination at 1 hour. For study termination animals were euthanized with pentobarbital overdose i.v. Following euthanasia the abdomen was cut open and the abdominal aorta and caudal vena cava were exposed and accessed with arterial sheaths. Consecutively the vessels were flushed with 500 ml heparinized Ringers solution via the arterial sheath until blood clearance. Treated external iliac arteries were then carefully dissected, explanted and snap frozen in liquid nitrogen. Subsequently the treated iliac arteries (n=4) were stored at 70 C. until shipment on dry ice to the analytic laboratory. At the laboratory explanted treated vessels were weighed, homogenized and the undiluted homogenate was measured for sirolimus content. At all times, the batch samples were clearly identified and were processed on the same day using the same extraction method. All undiluted samples showed over detection range sirolimus content and were repeatedly measured after being diluted 1:10 and 1:20.

(61) TABLE-US-00019 TABLE 18 Scheme of Ballon Dilatation Example 17 Animal Vessel Number Animal ID Time point Left iliac artery Right iliac artery 1 7_12 1 hour SIR 3 No. 27 SIR 3 No. 26 2 8_12 1 hour SIR 3 No. 28 SIR 3 No. 29
Results:

(62) Animals showed no sign of toxicity after sirolimus eluting balloon deployment and post expansion angiography revealed patent vessels and no sign of vessel wall dissection. Macroscopically, at the time of vessel explantation, there were also no signs of vessel trauma or dissection. The HPLC-based results show that there was remarkable sirolimus uptake into the vascular wall resulting in a mean concentration of 35.0033.37 ng/mg. The tissue sirolimus concentrations ranged from 8 to 82 ng/mg.

(63) TABLE-US-00020 TABLE 19 Tissue sirolimus content per treated vessel Tissue sirolimus Balloon to concentration Group Device Number artery ratio Time point (ng/mg) SIR 3 No. 26 1.1:1 1 hour 36.13 SIR 3 No. 27 1.1:1 1 hour 81.68 SIR 3 No. 28 1.1:1 1 hour 14.10 SIR 3 No. 29 1.1:1 1 hour 8.09

CONCLUSION

(64) The current study aimed to examine tissue concentrations of sirolimus 1 hour after sirolimus eluting balloon deployment. The currently applied sirolimus eluting balloon resulted in significant sirolimus concentrations within the treated arteries. To the best of our knowledge this is the first sirolimus-eluting balloon capable to achieve arterial wall tissue concentrations of maximal 82 ng sirolimus/mg tissue. In this regard, the applied carrier formulation of shellac and omega fatty acids is a promising new coating technology for the delivery of sirolimus to the arterial tissue.